Size-variable strain-specific protective antigen for potomac horse fever

ABSTRACT

An isolated and purified antigen which is expressed by a wild-type  E. risticii  strain and is specific to the strain. The present invention also relates to nucleic acid constructs which encode the antigen, expression vectors, transformed host cells, and methods for producing the antigen.

[0001] This application claims the benefit of priority under 35 U.S.C.§119(e) to U.S. Provisional Application Serial No. 60/059,252, filed onSep. 18, 1997.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to isolated and purified antigenwhich is expressed by a to wild-type E. risticii strain and is specificto the strain. The present invention also relates to nucleic acidconstructs which encode the antigen, expression vectors, transformedhost cells, and methods for producing the antigen.

[0004] 2. Discussion of the Background

[0005] Potomac horse fever (PHF), also known as equine monocyticebrlichiosis (EME), is an acute infectious disease of horses. PHF wasinitially recognized in 1979 in areas along the Potomac river inMaryland and Virginia. The causative agent was subsequently identifiedin 1984 as Ehrlichia risticii, an obligatory intracellular rickettsialorganism. Since then, PHF cases have been reported in many states of theU.S. and some provinces of Canada. Serological evidence suggests thepresence of E. risticii in parts of Europe and Australia. The maindisease features of PHF are fever, leukopenia, depression, anorexia anddiarrhea Some affected horses may also develop, colic or laminitis. Themortality is as high as 20-25% Recently, abortions in pregnant marescontracting the disease have been documented. PHF occurs mostly in thesummer months. Although most of the rickettsial pathogens aretransmitted by arthropod vectors and the seasonality of PHF alsosuggests this, all attempts to reveal the mode of transmission of E.risticii have been unsuccessful.

[0006]E. risticii infection is responsible for substantial economic lossto the equine industry. Currently, inactivated vaccines for PHF arecommercially available from three different manufacturers. In endemicareas, vaccination of equine population against PHF is performed on aregular basis. Despite this, PHF is occurring in increasing numbers,including in vaccinated horses. In 1990, E. risticii was isolated from ahorse suffering from severe PHF in spite of carrying a high titer ofantibodies from multiple PHF vaccinations. On Western blot analysis, theantigenic profile of this newly isolated organism (90-12 strain) wasconsiderably different from that of the original organism (25-D strain)isolated in 1984 during the initial outbreaks of the disease. Insubsequent years, more isolates were obtained from vaccinated horsessuffering from clinical PHF. These findings suggested the possibleexistence of strain variation in E. risticii and its probable role invaccine failures in the field.

[0007] In the last few years, significant progress has been made towardunderstanding the pathogenesis and host immune response in E. risticiiinfection. Certain strains of mice have been identified to be goodlaboratory models of PHF. Various serological and DNA based tests havebeen developed to better facilitate diagnosis of the infection. Studiesto identify the antigenic composition of the organisms and the majorsurface antigens involved in immune response were conducted. However,most of these studies have been performed with the original E. risticiiisolates (isolated during 1984-85) from different laboratories. Exceptfor one recent report on biological diversity in E. risticii isolates,no systematic comparison between different isolates has been made toidentify the extent and importance of strain variation in this organism.Also, very little is known about the molecular biology of E. risticii.Hence, the present study has been undertaken to: i) understand thedifferences between the 25-D and 90-12 strains of E. risticii; ii)investigate the molecular basis of these differences; iii) identifyprotective antigen(s).

[0008] In addition to the main focus of problem solving E. risticiiinfections, there is an important scientific interest in these studiesto gain more knowledge on ebruichial organisms in general. Along with E.risticii, genus Ehrlichia of the family Rickettsiaceae contains someother recently identified organisms. New members of this genus includeE. chaffeensis and E. ewingii. pathogens of human and dog, respectively.Recently identified human granulocytic ehrtichiosis (HGE) has beendemonstrated to be caused by an organism similar to or the same as E.equi, an equine pathogen. Also, E. risticii has been found to infectdogs and cats. Emergence of these ehrlichial diseases and changes inhost specificity of ehrlichial organisms are quite intriguing.Information on the important proteins of E. risticii and the genes theyare encoded by may provide us with necessary clues to understand thesophisticated intracellular survival strategies of ehrlichial organismsand the natural dynamics in their ecosystem that lead to changes intheir life cycles.

SUMMARY OF THE INVENTION

[0009] The present invention is based on the discovery that strains ofEhrlichia risticii express surface antigens that are specific to thestrain. These surface-expressed proteins are termed strain-specificantigens (SSAs). These antigens have now been isolated and purified fromthe respective strains. The SSAs of the present invention may be used todetect Ehrlichia risticii strains and to generace a protective immuneresponse against E. risticii, strains, leading to the development ofmore effective vaccines against PHF.

BEEF DESCRIPTION OF THE DRAWINGS

[0010] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings.

[0011]FIG. 1: Primers sequences ECP 1 and ECP 2 for amplifying any SSAgene (SEQ ID NO: 1 and 2).

[0012]FIG. 2: Nucleotide sequence of 85 kD gene with flanking regionsand deduced amino acid sequence of the strain-specific antigen from E.risticii 90-12 strain (SEQ ID NO: 3 and 4). Putative −10, −35. RBSregions are underlined and putative starts of transcription is denoted(+1). The dyad symmetry and the adjacent thymine-rich regions areunderlined.

[0013]FIG. 3: Nucleotide sequence of 50 kD gene with flanking regionsand deduced amino acid sequence of the strain-specific antigen from E.risticii 25-D strain (SEQ ID NO: 5 and 6). Putative −10, −35, RBSregions are underlined and putative starts of transcription is denoted(+1). The dyad symmetry, and the adjacent thymine-rich regions areunderlined.

[0014]FIG. 4: Nucleotide sequence of ATCC-50 kD gene with flankingregions and deduced amino acid sequence (SEQ ID NO: 7 and 8). Putative−10, −35, RBS regions are underlined and putative starts oftranscription is denoted (+1). The dyad symmetry, and the adjacentthymine-rich regions are underlined.

[0015]FIG. 5: Analysis of deduced amino acid sequences of SSA homologuesfrom two antigenic variants of E. risticii (SEQ ID NO: 4 and 6). Therewere a total of eight identical domains present in both 50 kD and 85 kDantigens. The number at the top show each identical domain. There weresignificantly high homology present in the corresponding domain of thesame number. Minor amino acid changes in each domain in 85 kD identifiedafter compared with 50 kD and marked by a black triangle head.

[0016]FIG. 6: Pre-challenge serum antibody titers of mice from differentgroups of experiment 1. All the antigens used for immunization of micewere from the 90-12 strain. Antibody titers were determined byperforming indirect immunofluorescent assay (IFA). MM cells infectedwith the 90-12 strain were used in the IFA.

[0017]FIG. 7: Pre-challenge serum antibody titers of mice from differentgroups of experiment 2. Antibody titers were determined by performingIFA using MM cells infected with the 90-12 strain.

[0018]FIG. 8: Post-challenge clinical signs of mice from differentgroups of experiment 2. Clinical signs were scored on a scale of 0 to 5,with 5 representing the most severe symptoms.

[0019]FIG. 9: DAF patterns (size of the amplified DNA in ethidiumbromide agarose gel electrophoresis) of field strains of E. risticii.Group 1 (1.,8 kb): Isolates 94-2, 94-3, 94-24, 90-30 and 25-D strain inlanes 3, 4, 5, 7 and 2. Group 2 (1.86 kb): Isolate 94-27 in lane 6.Group 3 (1.80 kb): Isolate 94-28 in lane 13. Group 4 (1.75 kb): Isolates94-8, 94-31, 94-37, 94-49 and 94-50 in lanes 12, 13, 15, 16 and 17 whichis similar for Illinois/ATCC strain (1.75 kb). Group 5 (1.56 kb):Isolate 64--29 and 90-12 strain in lanes 11 and 8. Group 6 (1.45 kb):Isolates 94-22 and 94-25 in lanes 9 and 10. The DNA from uninfectedmouse macrophage cells were used as a control in PCR amplification (lane18). No visible band in lane 18 indicates the specificity of theprimers. Molecular weight markers in lanes 1 and 19.

DETAILED DESCRIPTION OF THE INVENTION

[0020] As used herein, the term “isolated and purified” refers to anantigen that has been separated and isolated from the E. risticii strainexpressing the same. Preferably, the inventive SSA is separated fromother proteins derived from E. risticii, especially other antigenicproteins.

[0021] The SSAs of the present invention may be obtained via PCRamplification from the genomic DNA of a wild-type E. risticiistrainusing well-known molecular biology techniques. Such techniques arewell-known to those skilled in this art. The oligonucleotide primers forisolating a desired SSA gene may be prepared based on the specificnucleotide sequences disclosed herein. Specific examples of suitableprimers are shown in FIG. 1 (SEQ ID NO: 1 and 2). For a discussion ofPCR amplification, see Current Protocols in Molecular Biology, F. M.Ausubel et al, Eds., Volumes 1-3, John Wiley and Sons, 1998,incorporated herein by reference.

[0022] The SSA may vary widely in both overall size and amino acidcomposition. The SSA may have a molecular weight of about 40 to about 90kDa, inclusive of all specific values and subranges there between. Inspecific embodiments of the present invention, the SSA has a molecularweight of about 50 kDa or 85 kDa. Examples of specific amino acidsequences of the inventive SSAs are shown in FIGS. 2-4 (SEQ ID NO: 3, 5and 7).

[0023] The present invention also provides isolated and purified nucleicacids (e.g., recombinant DNAs) which encode the SSAs. Specific examplesof nucleotide sequences encoding the SSA of the present invention areshown in FIGS. 2-4 (SEQ ID NO: 4, 6 and 8). All nucleotide sequencesencoding a particular SSA are included in the scope of the presentinvention. Selecting a nucleic acid encoding a particular amino acidsequence may be readily accomplished using the well-established geneticcode relating the nucleic acid sequence of a codon sequence to the aminoacid sequence encoded thereby. The genetic code is provided by R. H.Abeles et al, Biochemistry, Jones and Bartlett, 1992, p. 269,incorporated herein by reference in its entirety.

[0024] All percentage identities for the amino acid and DNA sequencesnoted above can be determined using a variety of algorithms known in theart. An example of a useful algorithm in this regard is the algorithm ofNeedleman and Wunsch, which is used in the “Gap” program by the GeneticsComputer Group. This program finds alignment of two complete sequencesthat maximizes the number of matches and minimizes the number of gaps.Another useful algorithm is the algorithm of Smith and Waterman, whichis used in the “BestFit” program by Genetics Computer Group. Thisprogram creates an optimal alignment of the best segment of similaritybetween two sequences. Optimal alignments are found by inserting gaps tomaximize the number of matches using the local homology algorithm ofSmith and Waterman. It is preferred to use the algorithm of Needlemanand Wunsch to compare the amino acid and DNA percentage identity in thepresent case to another amino acid or DNA sequence.

[0025] The nucleic acid encoding the SSA may be incorporated into avector suitable for directing the expression of the SSA in a suitablehost (i.e., recombinant expression). Such expression vectors may haveall of the customary transcriptional control elements which enable theSSA to be expressed in a host transformed with the vector. For adetailed discussion of expression vectors and related cloningtechnology, see Current Protocols in Molecular Biology, supra.

[0026] Suitable host cells include bacteria customarily used in theoverproduction of recombinant protein sequences, e.g., E. coli.Mammalian cells may also be used as host cells if desired.

[0027] The inventive SSA may be produced by culturing 2 host celltransformed with an expression vector carrying the nucleic acid encodingthe antigen in a suitable culture medium. The antigen is then isolatedfrom the culture medium according to well-known procedures.

[0028] The isolated and purified SSA may be formulated into animmunogenic pharmaceutical composition by incorporating an effectiveamount of the antigen into a pharmaceutically acceptable carrier.Suitable carriers include, for example, aqueous solutions containing thecustomary components for administration to host, e.g., buffers, salts,adjuvants, etc. Upon administration of the composition to a host, theantigen induces a protective immune response against the E. risticiistrain from which the antigen was derived. Preferably, such animmunogenic composition is a vaccine against the wild-type E. risticiistrain from which the antigen was derived. Of course, in a preferredembodiment, the antigen also produces an immune response against otherstrains besides the wild-type strain from which the antigen is derived.In other words, a SSA from one strain may contain one or more epitopeswhich are shared with the SSA of other strains. A suitable host for theinventive immunogenic composition is, for example, a horse. The host maybe any other animal that is susceptible to infection by E. risticii(e.g., cats, dogs and humans). Formulating immunogenic pharmaceuticalcomposition, administering the composition to a host, and determiningthe level of induced immune response are readily accomplished usingtechniques well-known to those skilled in this art.

[0029] Having generally described this invention, a furtherunderstanding can be obtained by reference to certain specific exampleswhich are provided herein for purposes of illustration only and are notintended to be limiting unless otherwise specified.

EXAMPLES EXAMPLE 1 Isolation of Strain Specific Surface Antigen Gene ofEhrlichia risticii

[0030]Ehrlichia risticii strains and DNA preparation:

[0031] Two different strains of E. risticii were used for this study.The original E. risticii strain (25D) was isolated in 1984, during theinitial outbreaks of PHF near the Potomac River bank in Maryland andVirginia. Recently the inventors isolated a new strain of E. risticii(90-12) from a vaccinated horse suffering from clinical PHF. These twostrains of the organism were grown separately in human histiocyte cellsand purification was accomplished over linear Renografin (Squibb, N.J.)density gradient centrifugation.

[0032] Propagation of Ehrlichia risticii in Cell Culture:

[0033]E. risticii strains were propagated a human histiocyte (HH) cellline (American Type culture Collection #U937). These cells were grown inRPMI 1640 medium (Flow Laboratories, McLean, Va.), supplemented with 4mM L-glutamine (M.A. Bioproducts, Walkersville, Md.), and 15% fetal calfserum (Gibco Laboratories, Grand Island, N.Y.). Approximately 20×10⁶cells in the logarithmic phase of growth were centrifuged at 500×g for10 minutes and the cell pellet was resuspended in 20 ml of E. risticiiinfected HH cell culture. This infected cell mixture was dispensed intoa 150 cm² tissue culture flask and incubated at 37° C. in a humidifiedchamber in the presence of 5% CO₂ for one hour. Seventy ml of the growthmedium was then added and the culture was incubated further.

[0034] The infected cell cultures were examined for infection byacridine orange staining according to a standard procedure. For this,about one ml of the cell suspension was centrifuged at 500×g for fiveminutes. The cell pellet, resuspended in about 50 μl of the supernatant,was applied onto a glass slide and allowed to air dry. The cells werefixed with absolute methanol for 10 minutes, stained with acridineorange stain for three minutes and examined with an ultravioletmicroscope. The efficiency of infection of E. risticii was determined byconsidering the number and the intensity of orange specks of E. risticiiinside the pale green stained cytoplasm of the HH cells.

[0035] The infected cultures were harvested on day 4-6 postinfection,depending upon the observed levels of infection. Infected HH cultureswere centrifuged at 17,000×g for 20 minutes in a Sorvall refrigeratedcentrifuge (Sorvall, Norwalk, Conn.) and the cell pellet was resuspendedin sodium-potassium-glutamine buffer to a final concentration of 50× andstored at −70° C.

[0036] Purification of Ehrlichia risticii:

[0037]E. risticii organisms were purified by centrifugation over alinear Renografin gradient according to known procedures described. Tenml of 50× concentrate of infected HH culture were diluted with 20 ml ofTris buffer (10 mM Tris, pH 7.4). All the buffers contained 1.0 mMphenylmethylsulfonylfluoride (PMSF) and 1.0 mM iodoacetamide asproteinase inhibitors. The cell suspension was homogenized for threecycles in a Omni mixer (Dupont Co., Wilmington, Del.) at maximum settingfor 30 seconds. Each cycle was followed with 30 seconds of cooling onice. The homogenate was clarified at 2,000×g for 10 minutes to sedimentthe nuclear material and unbroken cells. The supernatant was centrifugedat 17,000×g for 20 minutes. The resulting pellet was resuspended in 2.0ml of Tris buffer and the suspension was forced through 18 and 23 gaugeneedles to obtain homogeneity. The volume was brought up to 12 ml withTris buffer containing 10 mM MgSO₄. Two μl (20 μg) of Dnase I (LifeTechnologies, Inc., Gaithersburg, Md.) were added to the suspension andincubation carried out at 37° C. for about 10 minutes to digest theliberated host nuclear material. Two ml of the suspension were thenlayered on about 34 ml of a 20 to 45% linear density gradient ofRenografin (Squibb Chemical Co., New Brunswick, N.J.) in TED buffer (50mM Tris, pH8.0, 25 mM EDTA, 0.9% NcCl). The gradients containingehrlichiae were centrifuged at 83,000×g for one hour at 4° C. Theehrlichiae were observed to band at a density of about 1.182 gm/ml andcould be visualized well with a pointed light source. The cellulardebris formed a compact band at the too of the gradient. The ehrlichialbands were collected and diluted with 10 volumes of Tris buffer andcentrifuged at 17,000×g for 20 minutes to remove the Renografin. Thepurified E. risticii pellet then was resuspended in TEN buffer to afinal concentration of 500× for the DNA experiments or to a finalconcentration of 200× in 10 mM tris buffer, pH7.4, for all the analyses.

[0038] Extraction of Elirlichia risticii DNA:

[0039] Purified E. risticii suspension (500×) in TEN buffer was treatedwith lysozyme (Sigma Chemical Co., St. Louis, Mo.) at a finalconcentration of 2.0 mg/ml and incubated in a 37° C. water bath for 30minutes. To this digest, SDS was added to a final concentration of 0.5%and the lysate was kept in a 65° C. water bath for an additional 30minutes. This lysate was then treated with proteinase K (BethesdaResearch Laboratories., Gaithersburg, Md.) at a Final concentration of400 mg/ml and was incubated in a 56° C. water bath overnight. Two phenolextractions were done with equal volumes of water saturated phenol andby shaking on a Orbitron rotator (VWR Scientific, Brisbane, Calif.) for30 minutes each. Three chloroform (chloroform:isoamyl alcohol; 24:1)extractions were done and the DNA was precipitated by the addition ofsodium acetate (pH 5.2) to a final concentration of 0.3M. Two volumes ofabsolute ethanol was added and incubation was carried out at −20° C. fortwo hours. The precipitate was pelleted by centrifugation at 12,000 rpmin a table top microcentrifuge for 15 minutes at 4° C. The DNAprecipitate was washed once with 70% ethanol and then with absoluteethanol each followed by centrifugation at 4° C. The DNA pellet wasallowed to dry in a vacuum for five to ten minutes and then it wasdissolved in TE buffer (10 mM Tris, pH 8.0, 1.0 mM EDTA) to aconcentration of 1.0 μg DNA/μl and stored at −20° C. for future use.

[0040] Polyacrylamide Gel Electrophoresis and Western Immunoblotting:

[0041] Discontinuous SDS PAGE analyses were carried out over 10 and 12%polyacrylamide gels according to the method of Laemmli (120). Gels werecast on a vertical slab gel electrophoresis system (Model SE 600,Hoeffer scientific Instrument, San Francisco, Calif.). For a 10% gel, 10ml of acrylamide solution containing 30% acrylamide and 2.7%N,N′-methylene bisacrylamide were mixed with 7.5 ml of 1.5M Tris buffer,pH 8.8, 150 μl of 20% SDS, and 10.5 ml of distilled water and degassedfor 15 minutes under vacuum. Polymerization was initiated by theaddition of 150 μl of 10% ammonium persulfate and 10 μl TEMED(N,N,N′,N′-tetramethylethylene- diamine), and then the solution waspoured immediately into the gel apparatus. About 1.0 ml of watersaturated butanol was layered on top and the gel was allowed topolymerize for about 30 minutes. The stacking gel contained 1.33 ml ofacrylamide solution, 2.5 ml of 0.5M Tris buffer, pH 6.8, 100 μl of 10%SDS, 6.1 ml of water, 50 μl of 10% ammonium persulfate and 5.0 μl ofTemed. The samples were dissolved in sample buffer (62.5 mM Tris, pH6.8,2% SDS, 5% 2-mercaptoethanol, 10% glycerol) and heated to 100° C. for 10minutes, and loaded onto the gel. The gels were electro-phoresed for1111 volthours at a constant current with an automated power supply(model 3000 xi, Bromo-Pad laboratories. Riclunond, Calif.) while theapparatus was kept cooled to 4° C. using a thermostatic circulator (LKBInstruments, Bromma. Sweden). The gels were stained with 0.05% Coomassieblue in 40% methanol, 10% acetic acid, or processed for Westernblotting.

[0042] The Western immunoblotting was conducted according to the methodof Towbin et al. using a transfer apparatus (Hoffer). The Westernblotting sandwich contained 3.0 mm Whatman filter paper (WhatmanLimited, England), nitrocellulose membrane (NCM; Bromo-Rad),polyacrylamide gel, and 3.0 mm filter paper in that order. The sandwichwas assembled in a tray containing blotting buffer such that no airbubbles were trapped between the sandwich layers. The transfer wasperformed at 100 volts for four to six hours with a transfer powersupply (Hoffer). The temperature was maintained at 4° C. during thetransfer using a thermostatic circulator.

[0043] After the transfer, the NCM were cut, using the pre-stainedmolecular weight marker (Bromo-Rad) lane as a guide, and the unboundsites were blocked by incubation in a to percent casein solution forthree hours at 4° C. The antibodies were diluted in two percent caseinsolution and incubated with the membranes in a 150 mm diameter petridishes or in hybridization bags (BRL) for three hours at roomtemperature, or for overnight at 4° C. The membranes were washed twicein Tris saline (10 mM Tris, pH 7.4, 150 mM NaCl), with 0.05% TritonX-100, and once in Tris saline for 15 minutes each. The membranes werethen incubated with the appropriate alkaline phosphatase labeledantibodyies (Kirkegaard and perry laboratories, Inc.,) diluted to 2.0μg/ml in casein solution, for one hour at room temperature. Themembranes were washed as described earlier, followed by a final washwith 0.9% NaCl. Color development was accomplished with Fast Red TR saltand napthol AS MX phosphate substrates for about 10 minutes, and thereaction was stopped by washing the membrane in distilled water. Thediluted sera and enzyme-labeled antibody solutions were stored at −70°C. for reuse.

[0044] Cloning of Ehrlichia risticii Genomes of Original (25D) andVariant (90-12) Strains:

[0045] Fragments of the genomic DNA of E. risticii (25D strain) weremolecularly cloned in λ-gt11 vectors and a recombinant expressing acomplete 50 kD protein antigen gene was identified. Additional cloningof E. risticii (90-12 strain) was performed with similar procedures Inλ-ZAP (Stratagene, LA Jolla, Calif.) as described below

[0046] Construction of Variant Ehrlichia risticii Recombinants:

[0047] Restriction enzymes were obtained from Bethesda ResearchLaboratories (Gait-hersburg, Md.), Promega Corporation (Madison, Wis.)and New England Biolabs (Beve-rly, Mass.). T4 DNA ligase, λ packing mix,λ-ZAP II, pBluescript phagemids, and E. coli strain X11-Blue [recA1endA1 gyrA96 thi hsdR17 (r_(k) ⁻m_(k) ⁻) supE44 relA1 λ⁻ (lac) {F′ proABlac,⁴ Z M15, Tn10(tet^(r))}], were obtained from Stratagene (La Jolla,Calif.).

[0048] Restriction Enzyme Digestion of Ehrlichia risticii DNA:

[0049] Variant E. risticii genomic DNA was restriction digested by usingSau3AI (New England Biolabs) site-specific endonuclease in the followingmanner: Six μl of DNA sample containing 1.0 μg/μl of DNA were mixed with36 μl of distilled water and 5.0 μl of 1×Sau3A I digestion buffer [100mM NaCl, 10 ml Tris-HCL, 10 mM MgCl₂, (pH 7.3)], supplemented with 0.5μl (100 μg/ml) bovine serum albumin. The contents of the tube weregently mixed in an eppendorf centrifuge at 10,000 rpm for five seconds.Finally, 2.5 μl of enzyme (10 units/μl) were added and the mixture wasagain centrifuged at 10,000 rpm for five seconds in an Eppendorfcentrifuge, and was kept at 37° C. in a water bath for one hour. Thereaction was stopped by the addition of EDTA to a final concentration of25 mM. A small aliquot was electrophoresed over 1% agarose gel tomonitor the digestion. One hundred μl of TE buffer were added to themixture and the DNA was extracted once with phenol and subsequentlywashed three times with chloroform:isoamyl alcohol at the ratio of 24:1,The restriction digested DNA was precipitated with ethanol as describedabove.

[0050] Synthesis and Ligation of Adapters to Ehrlichia risticii DNAFragments:

[0051] Three different types. (1, 2, and 3) of EcoR I-BamH I conversionadapters were prepared by the annealing of six different kinds ofsynthetic oligonucleotides, and each of these adapters was ligatedseparately to the Sau3A I cohesive ends of the variant E. risticii DNAfragments.

[0052] Synthesis of Duplex Oligonucleotide Conversion Adapters:

[0053] Each oligonucleotide used to form the duplex conversion adapterswas synthesized by and obtained from Oligos ET Inc. (Wilsonville,Oreg.). One strand (A strand) of each duplex conversion adapter containsthe EcoR I cohesive end (AATT) at the 5′ terminus to the 10 mer coreannealing sequence. Three lengths of the “A strand” (A1, A2, and A3)were synthesized by the addition of single cytosine residues between the5′ end of the core sequence and 3′ end of the EcoR I cohesive end.Oligonucleotides complimentary to each length of the “A strand” coreannealing sequences (14mer=B1, 15mer=B2, 16mer =B3) were synthesizedwith Sau3A I, Mbo I or BamH I cohesive termini (GATC) added to the 5′end of the “B strand”. The duplex conversion adapters were formed byseparately annealing “A strands” and “B strands” with matching lengthsof complimentary core sequences. For this, a 0.5 A₂₆₀ unit of each ofthe lyophilized oligonucleotide was dissolved in 120 μl of distilledwater to obtain a 50 μM solution. Forty μl of each of thesecomplimentary oligonucleotides (A1+B1, A2+B2, A3+B3) were mixed with 10μl of 10× buffer (250 mM Tris, pH 8.0, 100 mM MgCl₂) and 10 μl ofdistilled water. These mixtures were heated separately to 95° C. andslowly cooled (approximately one hour) to room temperature. This yieldeda 20 μM solutions of 1, 2 and 3 types of adapters. At this point thethree lengths of each duplex conversion adapters with identical cohesiveends were stored separately at −80° C. for future use.

[0054] Ligation of Adapters toEhrlichia risticii DNA Fragments:

[0055] Dried ethanol precipitate of Sau3A I E. risticii restrictionfragments (6 μg) was resuspended in 45 μl of distilled water and wasaliquoted in three equal parts. Next, 15 μl of preannealed adapters type1, 2 and 3 were added to parts 1, 2 and 3 respectively to yieldapproximately a 10:1 molar ratio of adapter to the insert fragments. Toeach of these mixtures, 5.0 μl of 10× ligase buffer (500 mM Tris, pH7.5, 70 mM MgCl₂, 10 mM DTT), 0.5 μl of 10 mM ATP, 13 μl of distilledwater, and 1.5 μl (6 Weiss units) of T4 DNA ligase (Stratagene. LaJolla, Calif.) were added, mixed well and incubated at 15° C. for sixhours. After completion of ligation reaction the contents of the threeEppendorf tubes were mixed together in one tube and were placed in a 70°C. water bath for 10 minutes to heat inactivated the liase enzyme.Subsequently the tubes were cooled on ice.

[0056] Phosphorylation of Adapter Modified Insert DNA and Removal ofExcess Adapters:

[0057] Adapter modified insert DINA was prepared for ligation into λ-ZAPvector (Stratagene, La Jolla, Calif.) by phosphorylation of adapter 5′ends with T4 polynucleotide kinase (Promega Corporation, Madison, Wis.)and removal of excess adapters by spin column chromatography. Followingheat inactivation and cooling, 150 μl of reaction mixture were added to20 μl of 10×T4 polynucleotide kinase buffer (500 mM Tris-HCL, pH 7.5,100 mM MgCl₂, 50 mM DTT, 1.0 mM spermidine), 10 μl of 0.1 mM ATP, 1.0 μlof T4 polynucleotide kinase (10 units), and 19 μl of distilled water.The reaction mixture was incubated at 37° C. for 30 minutes and reactionwas terminated by single extraction with 1 volume of TE-saturatedphenol, followed by three extractions of equal volume ofchloroform:isomyl alcohol (24:1). The upper aqueous phase wastransferred to a fresh tube and unligated adapters were efficientlyremoved with spin column chromatography

[0058] The Sephacryl S-400 matrix, spin columns, wash tubes andcollection tubes for column chromatography were obtained from PromegaCorporation (Madison, Wis.). The chromatography columns were preparedaccording to the instruction of the Promega technical bulletin (#067).Briefly, Sephacryl S-400 slurry was thoroughly mixed and 1.0 ml slurrywas transferred to a spin column. The column tip was placed in the washtubes and then the whole assembly was placed inside a large centrifugecube (Falcon #25319) and centrifuged in a swing bucket rotor at 800×gfor five minutes. The wash tube with fluid in it was discarded, and asecond centrifugation was performed in the same manner to discarded anyremaining fluid in the column. The phosphorylated reaction mixture withexcess adapters was applied to the top of the gel bed of the preparedcolumn and the column was placed into the collection tube. This wholeassembly was then centrifuged in the same manner as described before inthe column preparation step. The phosphorylated adapter modified insertDNA present in the eluant of the collection tube was then ethanolprecipitated at −20° C. overnight by adding 0.5 volume of 7.5M ammoniumacetate and 2.0 volumes of ethanol. The precipitated DNA was pelleted bycentrifugation at 4° C. for 15 minutes and the invisible pellet waswashed once with 70% alcohol prior to vacuum drying.

[0059] Ligation of Insert DNA to λ-ZAP Arms:

[0060] The adapter modified phosphorylated vacuum dried insert DNApellet was resuspended in 6.0 μl of TE (10 mM Tris, pH 8.0, 0.1 mMEDTA). The optimal vectorinsert ratio for efficient ligation wasobtained by aliquoting 2.5, 0.5 and 0.1 μl of the E. risticii insert DNAinto three separate tubes. One μg of EcoR I digested anddephosphorylated λ-ZAP arms (Stratagene) was added to each of the tubes,followed by 1.0 μl of 10× ligase buffer, 0.1 μl of 10 mM ATP, anddistilled water to 9.0 μl. Then 1.0 μl of T4 DNA ligase (4 Weiss units,Stratagene) was added and the solution incubated at 15° C. for sixhours.

[0061] Packaging of Recombinants λ-ZAP DNA :

[0062] In vitro packaging of λ-ZAP concatomers was done using thecommercially available packaging mix (Gigapack II Gold, Stratagene). Twoμl of concatamerized λ-ZAP recombinants were added to a freeze-thawextract tube. To this, 15 μl of sonicated extract were added and mixedwell. After a brief spin to pull the contents to the bottom, tubes wereincubated at 22° C. for two hours. This packaging reaction was stoppedby adding 500 μl of SM buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 8.0 mMMgSO₄, 0.01% gelatin) and 20 μl of chloroform. The reaction mixture wascentrifuged at 1500×g for five minutes to pull down the debris. Thesupernatant was transferred to another cube and 25 μl chloroform wasadded to it. This recombinant λ-ZAP stock was stored at 4° C. for futureuse in titrating and screening.

[0063] Titration and Amplification of the Recombinants:

[0064] Fifty ml of LB (Luria-Bertani) broth supplemented with 0.2%maltose and 10 mM MgSO₄ were inoculated with a single colony of XL1-Blue strain of E. coli. The cells were grown overnight at 37° C. in ashaking incubator. Then next day the cells were centrifuged at 1000×gfor 10 minutes, and resuspended in 25 ml of 10 mM MgSO₄ and stored onice. A 10-fold serial dilution of phage stock (packaging mix), up to10⁻¹⁰, was prepared in SM buffer and 10 μl aliquots from each dilutionwere mixed and incubated separately with 200 μl of above prepared hostcells. Each mixture was incubated at 37° C. for 15 minutes to absorb thephage on the surface of the host cells. Seventy ml of NZY top agar(0.75%) were equilibrated at 48° C. in a water bath; then 350 μl of 250mg/ml 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-gal) and 105 μlof 0.5M isopropyl-β-D-thiogalactoside (IPTG) were added to it. Seven mlof this molten top agar were mixed separately with each dilution ofphage-bacteria mixture and poured immediately onto a 100 mm petri dish.The plates were incubated at 37° C. for 6 hours and stored at 4° C.overnight for color development. The next day, the blue and clearplaques were counted to determine the titer and cloning efficiency ofthe packaged λ-ZAP.

[0065] To obtain a high titer library for storage, in vitro packagedrecombinants were amplified by plating approximately 50,000 plateforming units (pfu) and incubating at 37° C. for about 6 hours. Vixenthe places attained the size of about 0.5 mm, 10 ml of SM buffer wereadded to the plate and incubated overnight while shaking at 4° C. Thesuspension containing phage was extracted once with chloroform andstored in the presence of 0.3% chloroform.

[0066] Immunoscreening of Recombinants for Expression of VariantEhrlichia risticii Antigens:

[0067] The variant recombinant clones were screened for expression ofvariant E. risticii antigens using rabbit and mouse antisera against thevariant E. risticii strain. Before use, the rabbit antisera wasexhaustively absorbed against E. coli and λ-ZAP protein components.

[0068] Production of Antiserum Against Variant Strain:

[0069] Hyperimmune sera to the variant strain of E. risticii wereproduced in rabbits. The first injection contained 80 μg and 320 μg ofthe purified organism, emulsified with Freund's adjuvant, administeredby interdermal and intramascular routes, respectively. A secondinjection, administered two weeks later by the intramascular route,contained 200 μg of the purified E. risticii emulsified in Freund'sincomplete adjuvant. At tour and seven weeks following the firstinjection rabbits were again injected intramuscularly with 200 μg of theorganism only. One week after the final injection, sera were collectedand pooled. Antisera from mice infected or immunized with E. risticiiwere obtained as according to known procedures.

[0070] Absorption of Variant Ehrlichia risticii Antiserum:

[0071] Variant E. risticii antisera were exhaustively absorbed with thelysates of E. coli strain XL1-Blue and λ-ZAP phase to remove anynon-specific antibodies. An one Liter culture of XL1-Blue transformedwith pBluescript SK-phagemids (Stratagene) was grown in LB medium to anOD₆₀₀ of 0.5 at 37° C., and IPTG was added to 10 mM final concentration.The cells were harvested by centrifugation at 11,000×g for 10 minutesand the cell pellet was resuspended in 20 ml of 10 mM Tris, pH 7.5, 1.0mM phenylmethylsulfonyl fluoride (Sigma). About 15 ml of cell suspensionwere subjected to four 30 second cycles of sonication at 4° C. Next,Triton X-100 was added to 0.05% and the homogenate was incubated for 30minutes on ice and then diluted in 30 ml Tris saline (10 mM Tris, pH7.4, 150 mM NaCl), and stored at −70° C. This preparation of bacterialcell lysate was designated as sonic lysate. To the remaining 5.0 ml oforiginal cell suspension, Laemmli sample buffer (62.5 mM Tris, pH 6.8,2% SDS, 5% 2-mercaptoethanol, 10% glycerol) was added to 1×, heated to100° C. for five minutes, and diluted with 10 ml of Tris saline andstored at −70° C. This preparation of bacterial cell lysate wasdesignated as SDS lysate.

[0072] A large scale preparation of λ-ZAP phage particles was producedaccording to Maniatis. One Liter of X 11-Blue cells was grown up to theOD₆₀₀ =0.5, in LB media supplemented with 0.2% maltose and 10 mM MgSO₄.The culture was inoculated with 10¹⁰ pfu of phage particles andincubated at 37° C. for an additional five to six hours, until thevisible lysing of the bacterial cells was prominent as indicated bypresence of cell debris. The lysed culture was further incubated for 10minutes in presence of 20 ml of chloroform. Pancreatic DNAase-I andRNAase (Sigma), were added to this lysed culture to a finalconcentration of 1.0 μg/ml and a further incubation was performed for anadditional 30 minutes at room temperature. To disperse the phageparticles from the bacterial debris, 58.4 gm of solid NaCl were addedand the lysate was incubated at 4° C. overnight. The next day thebacterial debris was removed from this lysate by centrifugation at11,000×g for 10 minutes and 100 gm of solid polyethylene glycol (PEG8,000) were mixed into the supernatant. The mixture was incubated on icewater for 1 hour and the precipitated phage particles were recovered bycentrifugation at 11,000×g for 10 minutes. The supernatant was discardedand the phage pellet was resuspended in 20 ml of Tris saline and addedto the sonic lysate obtained earlier.

[0073] In separate polyethylene bags, ten 137 mm nitrocellulose circles(NCM, Schleicher & Schuell, Inc., Keene, NH) were incubated with soniclysate and another five membranes were incubated with SDS lysate for twohours at room temperature on a shaker. The membranes were then washedfive times with Tris saline for 15 minutes each and incubated overnightwith casein solution (2% casein in 10 mM Tris, pH 7.5, 120 mM NaCl) at4° C. Five ml of rabbit anti- E. risticii serum were diluted in 100 mlor casein solution and placed in a tray. Two NCM adsorbed with soniclysate and one NCM adsorbed with SDS lysate were placed in the tray andincubated for two hours. The membranes were taken out, replaced with newsets of membranes and incubated as before. The process was repeated withall the membranes. The absorbed serum was aliquoted and stored at −70°C.

[0074] Immunoscreening the Recombinants of Variant Strain:

[0075] Screening the λ-ZAP recombinants for expression of E. risticiiantigens was done according to known procedures. E. coli strain XL1-Blue was used as a host cell to plate the library. A liquid culturewas started from a single colony and grown overnight with vigorousshaking at 30° C. in LB media supplemented with 0.2% maltose and 10 mMMgSO₄. The cells were centrifuged at 1000×g for 10 minutes then gentlyresuspended in 0.5 volumes of 10 mM MgSO₄. About 700 to 1000 pfu of thepackaged λ-ZAP were mixed with 1.2 ml of above prepared XL 1-Blue cellsand incubated at 37° C. for 18 minutes. Twenty one ml of molten NZY topagar (0.8%), prewarmed to 42° C. were then added, mixed, and poured ontoa 150 mm place containing 1.5% NZY bottom agar and the agar was allowedto solidify at room temperature for 15 minutes. The places wereincubated at 37° C. for four hours, until the plaques were about one mmin size. Next, a 137 mm colony/pique screen membrane (NEN® Researchproducts, Boston, Mass.) was saturated with IPTG solution (10 mg/ml) andblotted dry on a filter paper. This membrane was carefully placed on thetop agar and incubation was continued at 37° C. for another three hours.The membrane was pierced asymmetrically at three places with an 18 gauzeneedle, peeled from the agar, and washed three times with Tris saline toremove the debris and bacteria. The plates were then stored at 4° C. andthe washed NEN membranes were blocked with casein solution at 4° C.overnight. The next day, membranes were incubated in a 1:100 dilution ofthe absorbed E. risticii antisera for two hours at room temperature andwashed twice in Tris saline with 0.05% Triton X-100, and once in Trissaline for 15 minutes each. The antisera treated membranes wereincubated either with 2.0 μg/ml of alkaline phosphatase labeled goatanti-rabbit IgG or mouse anti-rabbit IgG (Kirkegaard and Perry) for onehour at room temperature. The membranes were consecutively washed threetimes in the same way described earlier in tics procedure, followed by afinal wash with 0.9% NaCl. Finally the membranes were treated with FastRed and naphthol substrate solution for about 10 minutes and thereaction was stopped by washing the membrane in distilled water.

[0076] The pink immunoreactive spots correspondding to the recombinantsexpressing E. risticii antigens were aligned with the help of the needlemarks and those positive plaques were picked up from the plates with theaid of a Pasteur pipette. The agar plugs containing the recombinantplaques were dispensed separately into 500 μl of SM buffer and the phagewere allowed to diffuse out by vortexing and incubating vials at 4° C.for two hours. Twenty μl of chloroform were also added separately ineach vial before Long term storage. Plaque purification of therecombinants was accomplished by two additional rounds ofimmunoscreening as above.

[0077] Identification of Recombinant Antigens of Variant Strain:

[0078] The identity of the recombinant antigen expressed in the clonesor the λ-ZAP library was established by preparing monospecificantigen-selected antibodies and reacting this with the nitrocellulosestrips containing electrophoretically separated E. risticii antigens

[0079] Production of Recombinant Clone Specific Antibody:

[0080] The plaque purified λ-ZAP recombinants (10⁵ pfu) were mixedseparately with 1.2 ml of pre-prepared MgSO₄ treated XL 1-Blue cells andincubated at 37° C. for 18 minutes to absorb the phage on surface ofbacteria. Each phage bacteria mixture was plated on a 150 mm petri dishas described above. After the plaques had attained the size of 1.0 mm, a137 mm NCM saturated in IPTG solution (10 mg/ml) was overlaid on the topagar of the plate and incubated at 37° C. for four hours. The NCM wasreversed and incubation was continued for an additional three hours.After washing and blocking the unbound sites, as described above, theNCMs were incubated with the 1:100 dilution of antisera at 4° C.overnight. The membranes were washed once with Tris saline, twice inTris saline with 0.05% Triton X-100, and once in Tris saline for 15minutes each. The membranes were then placed separately in polyethylenehybridization bags (BRL) and 10 ml of glycine buffer (0.2M glycine, pH2.8, 150 mM NaCl) were added to each bag. The bags were heat sealed andincubated at room temperature for one hour to elute the antibodies. Theeluted antibodies were neutralized to pH 7.0 with 500 μl of 132M Trisbase and stored at −70° C. A preparation made from the non recombinantλ-ZAP was processed in same way as the negative control.

[0081] Identification of the Recombinant Antigens:

[0082] The recombinant clone-specific antibodies were diluted with anequal volume of casein solution. These antibodies were incubatedovernight at 4° C. with a strip of NCM on which electrophoreticallyseparated E risticii proteins had been blotted. Next, the strips weretreated with alkaline phosphatase labeled anti-rabbit IgG andsubstrates. The strips were now aligned with an adjacent strip which hadbeen reacted with polyclonal E. risticii antisera and the identity ofthe antigen encoded by the recombinant was ascertained.

[0083] Antigenic Analysis of Standard and Variant Strains:

[0084] Western immunoblotting was performed on both standard and variantE. risticii strains with their homologous and heterologus mouse antiseraby the procedure described above. Antigenic analysis of the componentantigens of these two different strains of E. risticii was alsoperformed by Western blotting with clone specific antibodies of 85 kD 55kD, 51 kD and 28 kD proteins of the variant strain and the 50 kD of thestandard strain. In order to perform this renograffin purified E.risticii of standard and variant strains were gel electrophoresed inseveral lanes in alternate combination. After blotting the NCM stripswere cut in such a way that each strip contained the antigens of boththe standard and variant strains. These strips were then treatedseparately with a clone specific antibody to determine the antigenicdifference and similarities between the strains. The techniques whichwere followed here were described above.

[0085] Construction of DNA Probe and DNA Hybridization:

[0086] The random prier labeling technique was used to incorporate theradioactive ³²P in E. risticii insert DNA of several recombinants. Thelabeled probes generated in this manner were used in Southernhybridization of the restricted E. risticii genome of standard andvariant strains.

[0087] Probe DNA:

[0088] The probe DNAs were prepared by using two different techniques.One of the techniques involved restriction digestion of the recombinantphagemids, agarose gel electrophoretic separation of the insert DNA, andelution of the insert DNA band from the gel. In this process, after getelectrophoresis, the agar piece containing the specific DNA band wasvisualized on an ultraviolet light trarsilluminator (Hoeffer, SanFrancisco, Calif.) and separated out from the gel by using a razorblade. The DNA Was extracted from the agarose gel piece by using silicabeads (GenecleanII, Bio101, La Jolla, Calif.), following themanufacturer's suggested protocol. Briefly the gel piece was weighed andabout three volumes of 6.0M sodium iodide were added and incubated at55° C. until the agarose completely dissolved. To this, 5.0 μl of silicabeads (Glassmilk, Bio101) were added and the emulsion incubated at roomtemperature for five minutes while occasionally mixing the silica beadswith the dissolved arose by tapping the tube. The silica beads wereseparated out from the solution by quick centrifugation in a table topmicrocentrifuge and washed with 10 mM Tris, pH 7.5, 100 mM NaCl, 1.0 mMEDTA, 50% ethanol (New Wash; Bio101). The process was repeated for twomore times and finally the DNA bound with the silica beads was eluted byresuspended the beads with 10 μl of distilled water and incubating at55° C. for five minutes.

[0089] The other technique involved PCR amplification of a segment ofDNA directly from the genomic DNA of E. risticii. Specific sequencesobtained from the cloned E. risticii recombinants were used to selectthe proper primer pair for each amplification. Typically a 100 μl PCRreaction mixture consisted of 10 μl of 10× reaction buffer (0.5M KC1,0.1M Tris, pH 8.3, 15 mM MgCl₂ and 0.1% gelatin), 16 μl ofdeoxyribonucleotide triphosphates (160 nmoles each), 4.0 μl of primers(0.1 nmole each), 0.5 μl of Taq polymerase (5units/μl), 10 μl of E.risticii genomic DNA (1 ∥l/ml) and 59.5 μl of distilled water. Theamplification was performed using a DNA Thermal Cycler (Perkin ElmerCetus, Norwalk, Conn.). The initial template denaturation step proceededfor 1.5 minutes at 95°πC. Then a typical cycle profile consisted ofannealing for two minutes at 52° C., extension for three minutes at 72°C. and denaturation for one minute at 94° C. A total of 60 cycles wereperformed. At the end of the 60th cycle the heat denaturation step wasomitted and the extension step was extended by an additional sevenminutes. Following the termination of the amplification cycle, thesample was allowed to return to at 4° C. temperature and held there. Thespecificity of the PCR amplified DNA was further confirmed by gelelectrophoresis and the DNA was purified by Geneclean II, following theprocedure described above.

[0090] Labeling of Probe DNA:

[0091] The random primer labeling was done by Prime a Gene® LabelingSystem (Promega Corporation). Twenty ng of DNA were diluted in 25 μl ofdistilled water and boiled at 100° C. for two minutes. The solution wasimmediately chilled on ice. With the tube held on ice, 10 μL of 5×labeling buffer (250 mM Tris-HCl, pH 8.0. 25 mM MgCl₂, 10 mM DTT, 1 MHEPES. pH 6.6, and 26 A₂₆₀ units/ml random hexadeoxyribonucleotideprimers), 2.0 μl mixture of nonlabeled deoxyribonucleotides (0.5 mM eachof dATP, dGTP, and dTTP), 2.0 μl nuclease free BSA (400 μg/mM 5.0 μl orα³² P (50 μCi) dCTP and 5.0 units of DNA polymerase I Klenow fragmentwere added. The mixture was incubated overnight at room temperature andfinally the labeling reaction was terminated by heating at 100° C. fortwo minutes. The unincorporated nucleotides from the reaction mixturewas removed by using the Push column (Stratagene) and the mixture wasstored at −20° C. for future use in a Southern hybridization cocktail.

[0092] The amount of incorporated radioactivity and the specificactivity was measured by TCA precipitation (10% trichloroacetic acid and1% sodium pyrophosphate). Specific activities in the range of 10⁹counts/minutes (CPM) per μg of DNA were obtained by the random primerlabeling method.

[0093] Southern Blot Hybridization:

[0094] The gel containing the DNA samples was acid-depurinated with0.25M HCl for 15 minutes, denatured with 0.4M NaOH-0.6M NaCl for 30minutes, and neutralized with 1.5 M NaCl/0.5M Tris, pH 7.5, for 30minutes. In preparing for capillary transfer, two layers of 3.0 mmWhatman filter paper were spread on the Plexiglas support of theblotting apparatus (BRL), and placed in a buffer tray filled with 10×SSCand a pipette was rolled over them to remove air bubbles. The gel wascarefully inverted and placed on the filter paper, and again the pipettewas rolled over to remove any trapped air bubbles. This whole assemblywas then covered with Saran-wrap and the plastic was cut exactly to theoutline of the gel. The Saran-wrap used to avoid direct contact betweenthe wick and the stacking paper towels, and it also helped to preventthe unnecessary evaporation of the buffer. Genescreen Plus Membranes(NEN products) were presoaked in distilled water for one minute and in10×SSC for 15 minutes. The membranes were placed on the gels and airbubbles were removed by rolling the pipette as before. Three sheets of3.0 mm Whatman filter paper were soaked in 10×SSC and placed on theblotting membrane and a 5″ stack of paper towels were laid on the top ofthese filter papers. The DNAs from the gel were capillary transferred tothe Genescreen Membrane overnight. The next day, the membrane wascarefully removed from the gel and was treated with 0.4 N NaOH for oneminute and 2.0×SSC/0.2M Tris, pH 7.5 for five minutes. Finally themembrane was placed on a 3.0 mm Whatman filter paper and DNAs werepermanently bound with the membrane by a 30 seconds exposure in an UltraViolet Crosslinker from Stratagene.

[0095] The membrane blot was prehybridized in a polypropylene bag for1.5 hours at 45° C in a prehybridization solution consisting of 8 ml ofHybrisol I (Oncor, Gaithersburg, Md.) and 2.0 ml of Hybrisol II (Oncor)solution. The probe DNA was denatured by boiling for two minutes andadded to a final concentration of 3×10⁶ counts/minutes (CPM) per ml ofprehybridization solution. The hybridization was continued at 45° C. for18 hours and then membrane blot was carefully removed from the bag. Themembrane was washed twice with 1.0×SSC, 0.1% SDS at room temperature for20 minutes each and once with 0.1×SSC, 0.1% SDS at 60° C. for one hour.The wet membrane was sealed in a hybridization bag and exposed to X-omatfilm (Kodak, Rochester, N.Y.) at −70° C. for varying time intervals. Themultiple rehybridization of the same membrane blot was also accomplishedby stripping the probe from the membrane. To do this the membrane wasboiled for 30 minutes in a solution of 10 mM Tris-HCl, pH 8.0, 1 mM EDTAand 1% SDS. The DNA molecular weights in a Southern blot were determinedby hybridizing the 1 kilobase DNA ladder (BRL) run in the adjacent laneof the gel with the ³²P labeled probe of one kilobase DNA ladder.

[0096] Recombinant DNA Procedures and Sequencing:

[0097] The variant E. risticii antigens were expressed by several λ-ZAPrecombinants. The in vivo excision of those λ-ZAP recombinants yieldedpBluescript SK(−) phagemid clones. The specific clones obtained from therecombinants expressing 85 kD antigen were further subcloned to obtainthe complete nucleotide sequence of the 85 kD gene. The λ-gt11recombinant of 50 kD antigen gene was cloned in pBluescript SK(+)phagemid.

[0098] In vivo Excision of pBluescript SK(−) Phagemid:

[0099] In vivo excision of the pBluescript SK(−) phagemids from theλ-ZAP recombinant phages was done according to the procedures of themanufacturer (Stratagene). The ExAssist™ helper phage and Solr™bacterial strain [el 4(mrcA), Δ(mcrCB-hsdSMR-mrr) 171, sbcC, recB, recJ,umuC∵Tn5(kan^(r)), uvrC, lac, gyrA96, reiA1, thi-1, endA1, λ^(R), {F′proAB, lac,⁻⁴ Z M15} Su⁻ (non-suppressing)] were also obtained from theStratagene. After being plate purified three times, a single recombinantplaque was lifted from the agar plate and transferred into a sterilemicrofuge tube containing 500 μl of SM buffer and 20 μl of chloroform.The tube was vortexed and incubated at room temperature for two hours todiffuse the phage from the agar block into SM buffer. The titer of thisphage stock was 10⁶ pfu/ml. In a 50 ml tube, 200 μl of 0.5 OD₆₀₀XL1-Blue cells were mixed with 100 μl of phage stock and 1.0 μl ofExAssist helper phage and incubated at 37° C. for 15 minutes. Next, 3.0ml of 2×YT media were added and incubation was continued for another 2.5hours at 37° C. in a shaker incubator. In this incubation period, aco-infection of the recombinant λ-ZAP phagemid and the ExAssist helperphage proceeded in the same XL 1-Blue cells. As a final result the newlycreated recombinant pBluescript SK(−) phagemids were packed inside ofthe ExAssist helper phage and released from the bacterial cells. Oncethe phagemids were secreted, the remaining XL 1-Blue cells were removedfrom the mixture by heating the tube at 70° C. for 20 minutes. The heattreatment killed all the bacterial cells while the phagemid remainedresistant to the heat treatment. The heat inactivated mixture was thencentrifuged at 4,000×g for 10 minutes to pellet the cellular derbies andthe supernatant was stored at 4° C.

[0100] To rescue the phagemid, 10 μl and 0.1 μl volumes of packagedphagemid stock from above were mixed with 200 μl of 0.1 OD₆₀₀c Solrcells (E. coli) separately and incubated at 37° C. for 15 minutes. About10 to 50 μl volumes were plated onto LB plates containing 100 μg/ml ofampicillin, and incubated at 37° C. overnight. Since the Solr cells wereresistant to λ-ZAP recombinant, the colonies which appeared the next dayon the plates contained the pBluscript SK(−) double stranded phagemidwith the cloned DNA insert. The bacteria infected with helper phasealone could not grow because they did not contain the ampicillinresistant gene.

[0101] Extraction and Purification of Phagemid DNA:

[0102] Phagemid template DNA was prepared for sequencing and otherrecombinant work by a known method. The cell containing the phagemidswere grown in lightly capped 15 ml plastic screw cap tubes with 5.0 mlof LB broth containing 100 μg/ml ampicillin. The cultures were aeratedby mixing them in a shaker incubator at 37° C. overnight. The followingday 1.5 ml of the cultures were transferred to 1.5 ml microfuge tubesand centrifuged for two minutes. The supernatant was removed, anadditional 1.5 ml of culture was added, and the tubes were againcentrifuged for two minutes. The supernatant was removed as completelyas possible and cellular pellet was resuspended in 100 μl of an ice coldsolution of glucose/Tris /EDTA buffer (50 mM glucose, 25 mM Tris-HCl, pH8.0 and 10 mM EDTA). The tubes were incubated at room temperature forlive minutes. Cells were lysed by the addition of 200 μl NaOH/SDSsolution (0.2 N NaOH, 1% SDS), gentle mixing, and incubation at roomtemperature for 10 minutes. Neutralization of NiaOH and precipitation ofSDS and chromosomal DNA was accomplished by the addition of 150 μl of3.0M potassium acetate, pH 4.8 with gentle mixing for at least 30seconds. The contents were centrifuged for five minutes at 4° C. andsupernatants were transferred to fresh tubes, centrifuged another fiveminutes, and again transferred to new tubes, avoiding the carryover ofany precipitate. To these supernatants, 1.0 ml of ice-cold absoluteethanol was added and the nucleic acids were allowed to precipitate at20° C. for 30 minutes. The nucleic acid precipitates were collected bycentrifugation at 4° C. for five minutes, washed with 70% ethanol, andthe pellets were dried. The nucleic acids were resuspended in 20 μl ofTE buffer and the RNA was digested by the addition of 1.0 μg of RNAase-Aat 37° C. for 30 minutes. About 2.0 μl of this mini preparation DNA wereused for restriction digestion analysis.

[0103] Subcloning of 50 kD Recombinant of 25D strain:

[0104] The insert of 50 kD antigen gene of 25D strain identified fromthe λ-gt11 library, was re-cloned in pBluescript SK(+) for restrictionmapping. This insert-plasmid of recombinant pBluescript SK(+) wasrestriction digested and the fragments were subcloned in pBluescriptSK(−) vector for further analysis and sequencing purposes. The internalsegment of the insert was also PCR amplified and subcloned for the sameinterest.

[0105] The specific restriction digestion was obtained by the Hind IIIenzyme. For this, 1.0 μg of pBluescript SK(−) phagemid (Stratagene) wasdigested with Hind III, and the completeness of digestion wasascertained by agarose gel electrophoresis. The DNA was then extractedwith phenol:chloroform and resuspended in 1.0× calf intestinal alkalinephosphatase buffer (Promega, 50 mM Tris, pH 9.0, 10 mM MgCl₂, 1.0 mMZnCl₂, 10 mM spermidine). Dephosphorylation of the 5′ PO₄ groups wasaccomplished by digestion with 2.0 units of calf intestinal alkalinephosphatase (Promega). The enzyme was removed from the reaction mixtureby phenol-chloroform extraction and DNA was ethanol precipitated asbefore, with additional washing in 70% ethanol to remove thepyrophosphate ions. Finally the DNA pellet was resuspended in 10 μl ofTE buffer. About 2.0 μl of mini preparation DNA (1.0 μg) were mixed with4.0 μl of the appropriate 10× digestion assay buffer (Promega) and HindIII restriction endonuclease (Promega) at a final concentration of 1.5unit/μg DNA. After complete digestion for one hour at 37° C., 8.0 μl ofthe gel loading buffer (Appendix 5) containing a marker dye, were addedto the tube. The reaction mixture was electrophoresed on 1% agarose gelby a submerged horizontal gel electrophoresis apparatus (BRL). MarkerDNA (1 kilobase ladder, BRL) was electrophoresed simultaneously tomonitor and compare the run of the DNA samples. Upon completion of theelectrophoretic run, the migration pattern of the DNA bands was viewedwith a 302 nanometer ultraviolet transilluminator (Spectoline, ModelT.P.-302). The upper band consisted of plasmid DNA and the lower twobands consisted of insert DNA of the 50 kD antigen gene. The insertbands, as ascertained by electrophoretic migration, were cut out fromthe gel and processed for purification of DNA by GenecleanII (Bio101)silica matrix.

[0106] One μl of the prepared vector (0.1 μg) was mixed with twodifferent 10 fold dilutions or insert DNA to obtain a nearly optimalratio (2:1, insert:vector). To each of these reaction mixtures, 1.0 μlof 10 mM ATP and, 1.0 μl of 10× ligase buffer (Promega, 1.0× is 3.0 mMTris, pH 7.8, 10 mM MgCl₂, 10 mM DTT and 5.0 mM ATP), were added. Eachwas brought to a final volume of 9.5 μl with distilled water. The DNAends were ligated with two units of T4 DNA ligase (Promega) and thesolution were incubated overnight at 18° C.

[0107] The E. coli XL1-blue competent cells were transformed with theligated DNA by electro-transfaormation, using the Bio-Rad Gene Pulserapparatus. The competent cells were produced according the proceduredescribed in Pulse controller instruction manual (Catalog=65-2098) ofBio-Rad. One liter of LB broth was inoculated with 1/100 volume of afresh overnight culture and grown at 37° C. with vigorous shaking to anOD₆₀₀ of 0.6. The rapidly growing culture was cooled on ice for 30minutes and the cells were harvested by centrifugation at 4,000×g for 15minutes in 4° C. The pellet was washed two times with one liter of icecold distilled water and finally the pellet was resuspended in 3.0 ml of10% glycerol. The prepared cells were aliquoted and stored at −70° C.Just before the electro-transformation, the frozen cells were thawed onice and 40 μl of the cell suspension were added to 2.0 μl of ligationmix. After one minute incubation on ice, the mixture was transferredinto a cold 0.2 cm electroporation cuvette and pulsed with a timeconstant of four to five milli seconds with a field strength of 12.5kV/cm. Immediately 1.0 ml of prewarmed SOC medium (2% Bacto tryptone,0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose) was added to the mixture and then incubated at 37°C. for one hour in an orbital shaker. About 20 μl aliquots were platedon LB plates containing 100 μg/ml ampicillin, X-gal, and IPTG. Afterovernight incubation at 37° C., single white recombinant colonies werepicked up, grown in 5.0 ml of LB medium with 100 μg/ml of ampicillin andstored at −70° C. in 15% glycerol.

[0108] Due to the presence of the direct repeats, the central segment ofthe 50 kD antigen gene was PCR amplified and subcloned separately. Thisstrategy was followed to avoid the binding of sequencing primer at morethan one place in the entire length of the gene. To do this, 1.0 pg of50 kD recombinant plasmid DNA was PCR amplified following the standardreaction parameter described above. The amplified product wasascertained by electrophoretic migration and the product was cloned inpCR™ II vector (Invitrogen). Briefly 1.0 μl of PCR amplified product wasmixed with 5.0 μl of distilled water, 1.0 μl of 10× ligation buffer, 2.0μl pCR™ II vector and 4 units of T4 DNA ligase. The mixture wasincubated at 12° C. overnight E. coli One Shot™ competent cells(Invitrogen) were transformed with the ligated DNA according to themanufacturer's procedure. Fifty μl of competent cells were thawed on iceand 2.0 μl of 0.5 β-mercaptoethanol and 1.0 μl of ligation mix wereadded to it. After incubation on ice for 30 minutes, the cells were heatshocked by placing in a 42° C. water bath for 30 seconds and immediatelytransferred to ice for two minutes. The transformed cells werere-vitalized by adding 450 μl or pre warmed SOC media and shaking in aincubator at 37° C. for one hour. About 100 μl aliquots were plated onLB plates containing 50 μg/ml ampicillin and X-gal. After incubation at37° C. overnight, single white colonies were picked up, grown in 5.0 mlof LB medium with 100 μg/ml of ampicillin and stored at −70° C. in 15%glycerol.

[0109] Subcloning of 85 kD Recombinants of 90-12 Strain:

[0110] Two different recombinant phagemid clones expressing the 85 kDantigen gene were identified from λ-ZAP library of 90-12 strain. Afterin vivo excision the recombinant phagemids DNA were extracted and thesize of the inserts from these two specific clones were ascertained byHind III and Sau3A I restriction enzyme digestion. Several insertfragments from Sau3A I restriction digestion products were furthersubcloned in pBluescript SK(−) vector for sequencing purposes. The twoclones which expressed the 85 kD antigen gene did not cover the 5′ endof this gene. To clone the 5′ end and obtain the complete sequence, theupstream 5′ region of the 85 kD gene was PCR amplified directly from thegenomic DINA of 90-12 strain. The specific primers used for this purposewere selected from the 5′ upstream and middle of the 50 kD gene sequenceof 25D strain and the PCR product was cloned in pCR™ II vector. This wasaccomplished according to the procedure described above.

[0111] DLNA Sequencing:

[0112] The double stranded DNA was sequenced according to the Sangersdideoxy chain termination method using the Sequenase® Version 2.0 kit(United States Biochemical, Cleveland, Ohio). This method involved thein vitro synthesis of a DNA strand from a single stranded DNA templateusing a DNA polymerase. Synthesis was initiated at only one site wherean oligonucleotide prirmer annealed to the template. The synthesis chainreaction was terminated by the incorporation of a nucleotide analoguethat would not support continued DNA elongation (hence the name chaintermination). The chain terminating nucleotide analogues were the 2′, 3′dideoxynucleoside 5′-triphosphates (dd NTPs) which lackted the 3′-OHgroup necessary for DNA chain elongation. When proper mixtures of dNTPsand one of the four ddNTPs were used, enzyme catalyzed polymerizationwas terminated in a fraction of the chain population at each site wherethe ddNTPs were incorporated. Four separate reactions, each withdifferent ddNTPs, gave complete sequence information. A radiolabelednucleotide was incorporated during the synthesis, so that the labeledchain of various lengths were visualized by autoradiography, afterseparation by high resolution electrophoresis.

[0113] The polymerase ‘Sequenase®’ a modification of bacteriophage T7DNA polymerase (United States Biochemical), was used for sequencing. Theunique properties of Sequenase® are high processivity, low 3′ to 5′0exonuctease activity, and the efficient use of nucleotide analogues.These characteristics produce radioactive bands of more uniformintensity and less background radioactivity than those obtained whenusing a large fragment of E. coli DNA polymerase I or reversetranscriptase. Synthetic oligonucleotides (Oligos ETC Inc), specific forDNA clones at different restriction sites, were used as sequencingprimers. Template DNA, purified by minipreparation was first annealed tothe sequencing primer. Then DNA synthesis was carried out in two steps.The first step labeling and the second step resulted in the accuratetermination of DNA synthesis using the dideoxynucleotides. In the firststep, the primer was extended using a limiting concentration ofdeoxynucleoside triphosphates, including the radiolabeled dATP. In thisstep, virtually complete incorporation of labeled nucleotide occurredinto DNA chains which were distributed randomly in length, from severalto hundreds of nucleotides. In the second step, the concentration of allthe deoxynucleoside triphosphates were increased and a dideoxynucleosidetriphosphate was added. Processive DNA synthesis occurred until allgrowing chains were terminated by a dideoxynucteotide. At this stage,the chains were extended on an average of several dozen nucleotides. Thereaction was ultimately terminated by the addition of EDTA andformamide. This was followed by denaturation electrophgresis andautoradiography.

[0114] Annealing of Eemplate and Primer:

[0115] The miniprep, RNA-free double stranded plasmid DNA was firstdenatured by the alkaline denaturation method prior to annealing thesequencing primer with the target sequence. To do so, 8.0 μl of miniprepDNA was mixed with 9.0 μl of distilled water, 2.0 μl of 2M NaOH, 1.0 μlof 4.0 mM EDTA, and the mixture was incubated at 37° C. for 30 minutesin a water bath. The mixture was neutralized by adding 0.1 volume of 3Msodium acetate (pH 5.0) and the DNA was precipitated with three volumesof ethanol at −70° C. for 15 minutes. After washing the pelleted DNAwith 70% ethanol, it was redissolved in 7.0 μl of distilled water and2.0 μl of Sequenase® (United State Biochemicals) reaction buffer, and1.0 μl (3.0 ng) of the appropriate orimer was added. The mixture washeated to 65° C. for two minutes and then slowly cooled down to ambienttemperature over a period of 30 minutes. Once the temperature was below35° C., annealing was complete.

[0116] Labeling Reaction:

[0117] To label the DNA, a labelirag mix, (supplied with the kit) wasdiluted five fold with distilled water (2.0 μl of labeling mix and 8.0μl of distilled water) in a sterile Eppendorf tube. One μl of Sequenasewas diluted with 7.0 μl of ice cold TE buffer in another sterileEppendorf tube. To the Eppendorf tube containing 10 μl of annealedtemplate-primer, she following were added sequentially: 1.0 μl of 0.1Mditluothreitol, 3.0 μl of diluted labeling mix, 0.5 μl of dATP(10/μci/μl), and 2.0 μl of diluted Sequenase®. After mixing, the tubecontents were incubated for five minutes at room temperature.

[0118] Terrinartion Reaction:

[0119] Four Eppendorf tubes were labeled A, C, G and T. Two μl of eachtermination mix (supplied in Sequenase® kit) were placed in therespective tubes. The termination tubes were prewarmed to 37° C. for oneminute in a water bath. When the labeling reaction was completed, 3.5 μlof labeling mixture was transferred into each termination tube. Thecontents were mixed and incubated at 37° C. for 5 minutes in a waterbath. Following incubation, 4.0 μl of stop solution (95% formamide, 20mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol) were added to eachtube to stop the reaction. The contents of the tubes were mixedthoroughly and stored at −20° C. until ready to toad on the sequencinggel for electroohoresis.

[0120] Sequencing Gel Electrophoresis:

[0121] A Baserunner 200 Sequencing apparatus (Eastman Kodak Company,Rochester, N.Y.) was used for electrophoresis of the sequencing gel. Tocast the 6% polyacrylamide gel, two clean, Sigmacote (Sigma) treatedglass plates were assembled using a vinyl side spacer (0.4 mm) and 50 mlgel mixture (28.35 g urea, 10.5 ml 40% bisacrylamide, 6.75 ml 10×TBE, 26ml of distilled water, 675 μl of 10% ammonium persulfate, 18 μl ofTEMED) was poured into the gel mold. The flat edge of the shark-toothcomb (0.4 mm) was inserted between the plates to a minimum depth of 2.0to 3.0 mm. After overnight polymerization, the comb was removed and thenplaced again with it's teeth facing the gel sandwich.

[0122] Then each buffer chamber of the apparatus was filled withapproximately 500 ml of electrophoresis buffer (1×TBE). The gel waspre-electrophoresed for 30 minutes at a constant power of 60 watts,before loading the samples. The DNA samples from the dideoxy sequencingreactions were heated to 80° C. for two minutes and then transferred toice immediately prior to loading onto gel. The wells of the gel wererinsed out using a 10 ml syringe, attached with a 18 gauge needle toremove urea that had diffused out from the gel. Three μl of sample fromeach tube marked A, C, G and T were loaded onto the gel in the wells inthat order (left to right). After loading, the sequencing gel waselectrophoresed at 55 watts to generate enough heat to keep the DNAdenatured. The surface temperature of the glass plate was maintained atleast 50° C. during electrophoresis. About three hours later, when thelower marker dve reached the bottom of the gel, another 3.0 μl of eachsample were loaded into new wells in the same order and the gel waselectrophoresed at 52 watts for another two hours. After the sampleswere run, the upper glass plates were disassembled carefully and the gelwas soaked with 10% acetic acid and 12% ethanol until the xylene cyanoldisappeared. This was done to ensure that all the urea was removed fromthe gel. The gel was removed from the lower glass plate onto a supportof 3.0 mm Whatman paper and placed in a get dryer for two hours.

[0123] The dried gel was placed in a metal cassette which had aspring-loaded lid to hold the gel and the film in a close contact. Thegel was exposed to X-Omat™ (Eastman Kodak Company) 18×43 cm film indirect contact with the gel. After overnight exposure, the film wasremoved and developed by using an automatic X-ray developer.

[0124] Analysis of DNA and Deduced Amino Acid Sequences:

[0125] The DNA sequence analysis was done by IBI Pustell software (IBILimited, Cambridge, England). Using the program “Protein Coding RegionLocator” the open reading frame (ORF) of DNA sequences were acertained.This program combines several method for locating potential codingregions in a DNA sequence. The first method searches both strands of theDNA sequence, looking for regions between user-set start and stop codons(ORE). In prokaryotes it uses ATG for starts and termination codons forstops, and searches for all possible six reading frames. The secondmethod uses a statistical search (Fickett's Testcode) which looks forregions of DNA pith biased usage of codons. This measurement is madeover a window of bases which Fickert has shown must be at least 200 forgood results. The probability can be set (0.29, 0.40, 0.77 or 0.92) toconfirm that the region located is a real codina region. The high valueof 0.92 was used for this analysis to maintain a high stringencycondition. A combined test was performed to get a potential regionmeeting both criteria.

[0126] The amino acid sequence analysis was performed by usingPeptide-Structure and Plot-Structure programs (PepPlot). PepPlot waswritten by Drs. ivichael Gribskov and John Devereux of the GeneticsComputer Group, and it was available through National Institute ofdHealth (NIH, Bethesda, Md.). Peptide-Structure makes secondarystructure prediction for a peptide sequence. The predictions measure forantigenicity, flexibility, hydrophobicity and surface probability.Plot-Structure displays these predictions graphically. Using thisprogram the secondary structure of a protein was predicted according tothe Chou-Fasman method hydrophilicity according to the Kyte-Doolittlemethod and antigenic index according to the Jameson-Wolf method.

[0127] Expression of 50 kD and 85 kD Homoloaue Antigen Genes:

[0128] After the full sequence analyses of the 50 kD and 85 kD majorantigen genes, they were cloned separately by PCR in the expressionvector pRSET C (Invitrogen). The advantage of using this expressionsystem was that the foreign prokaryotic genes were expressed in highamounts by the bacteriophage T7 promoter present upstream of the clonedgenes. This high level of expression was facilitated by infecting the E.Coli cells with M13 phage which expressed T7 RNA polymerase. For thecloning of the 50 kD and 85 kD antigen genes, two primers, one at the 5′end of start site of the gene and the other at the 3′ end of thetermination site of the gene, were selected. While synthesizing theseprimers, a sequence containing one restriction enzyme site was addedeight bases upstream of the 3′ end of each primer. Different restrictionenzyme sites were added to the two primers so that the amplified productcould be cloned in the desired orientation in the multiple cloning siteof the vector. The expression of the cloned gene and purification of theexpressed protein was done according to the recommendations of themanufacturer (Invitrogen). For easy purification of the expressedprotein, a metal binding polyhistidine domain and a site forenterokinase cleavage were also added by the vector sequence to theamino-terminal of the recombinant protein. The expressed recombinantprotein could be purified by binding to a nickel (Ni²⁺) charged resinand the extra Ni²⁺domain could be cleaved using enterokinase.

[0129] PCR Amplification of 50 kD and 85 kD Genes:

[0130] The complete 50 kD and 85 kD genes were amplified separately fromthe genomic DNA of the original and variant strain of E. risticii byusing two modified primers, named as expression cloning primers E.C.P-1and E.C.P-2 (FIG. 1). The E.C.P-1 (^(5′) CAT AAA ATT TCT AAG ACG AAG GATCCC TAT GTC ^(3′)) was selected from the known sequence of bp upstreamof the first methionine codon of the genes. This 33 base primer wasmodified at base 21 and 22 position by the substitution of two A's in itoriginal sequence with two G's. In the same way, E.C.P-2 (^(5′) GAG AGAAAG TTC CCC GTG TGA ATT CTA GCT AGG ^(3′)) was selected from the knownsequence 69 bp downstream of the stop codon of the gene. This 33 baseprimer also was modified at base 21 by introducing another single baseA. Amplification of the complete genes (50 kD and 85 kD) by using thesetwo modified primers produced BamH I and EcoR I sites at the extreme 5′and 3′ end of the genes respectively.

[0131] PCR amplification was accomplished according to the standardprotocol described above. As a template, the genomic DNA of the originaland variant strains were produced directly from their respective cellculture materials by the PCR lysis method (9). E. riticii infected HHcells (1million) were harvested on day five to seven postinfection andthe cell pellet was frozen and thawed three times to rupture the cells.Then 1.0 ml of PCR lysing buffer [50 mM KCl, 10 mM Tris (pH 8.3), 2.5 mMMgCl₂, 0.1 mg/ml gelatin, 0.45% Nonidet P40 (Sigma Chemicals), 0.45%Tween 20 and 0.06 mg/ml K ] was added to the ruptured cells and themixture was incubated at 62° C. for one hour. Finally the mixture wasincubated at 95° C. for seven minutes to heat inactivate the protinase Kand stored at 4° C. Ten μl of this preparation were used as a templatefor PCR amplification.

[0132] Cloning the PCR Amplified Products in SK(−) Phagemid:

[0133] The PCR amplified 50 kD and 85 kD genes were cloned separately inthe multiple cloning region of the Sk(−) phagemid (Stratagene) forfurther sequence analyses. This extra step of cloning and sequencing wasaccomplished to confirm the correct amplification of the ORF prior todirectional cloning in the expression vector pRSET-C.

[0134] To accomplish this amplified product (100 μl) was elecrrophorsedin an agarose gel and the specific DNA band was purified by GenecleanII, following the procedure described above. Fifty μl of this purifiedDNA were mixed with 6.0 μl of NEBuffer three (New England Biolabs), 3.0μl of distilled water, and 10 units each of BamH I and EcoR I enzymes(New England Biolabs), and double digested at 37° C. for one hour. Thedigested product was further purified by Geneclean II and eluted in 25μl of distilled water. The SK(−) phagemid DNA (1.0 μg) was also doubledigested and purified separately by using the same technique and elutedin 25 μl of distilled water. The ligation of vector and insert DNA wasaccomplished by mixing 2.0 μl of double digested phagemid DNA with 4.0μl of double digested PCR amplified product, 1.0 μl of 10×ligase buffer,0.1 μl of 10 mM ATP, 1 μl (4 Weiss units) of ligase (Stratagene), and1.9 μl of distilled water and incubating the mixture at room temperaturefor 2.5 hours. After the ligation reaction, the E. coli XL 1-bluecompetent cells were transformed with 2.0 μl of the ligation mixture byelectro-transformation technique and the recombinant clones wereselected by plating the transformed cells on LB plates containing 100μg/ml ampicillin, X-gal, and IPTG. DNA from the recombinant clones wereextracted by the minipreparation technique and sequenced to confirm thecomplete ORF of the 50 kD and 85 kD genes.

[0135] Cloning the 50 kD and SMD) Gene in pRSET-C Expression Vector:

[0136] Insert DNAs from the SK(−) recombinant clones of the 50 kD and 85kD genes, which were confirmed by the sequence analyses, were used forfurther cloning in pRSET-C expression vector. The selected recombinantswere grown in 50 ml of 2×YT media containing 100 μg/ml of ampicillin andrecombinant phagemid DNA was extracted by minipreparation following thetechnique described above. For cloning, 1.0 μg of each pRSET-C plasmidDNA (Invitrogen) and recombinant phagemid DNA were taken separately anddouble digested with BamH I and EcoR I restriction enzymes in a 60 μlreaction volume. The reaction conditions were the same as describedabove. After the restriction digestion, both recombinant phagemid andpRSET-C plasmid DNA were electrophoresed in an agarose gel in separatelanes. The specific insert DNA bands and linear pRSET-C plasmid DNAbands were purified from the agarose gel by Geneclean II and elutedseparately in 10 μl distilled water. One μl of the prepared vector (0.1μg of pRSET-C) was mixed with two different 10 fold dilutions of insertDNA to obtain a nearly optimal ratio (2:1::insert:vector). To each ofthese reaction mixtures, 1.0 μl of 10 mM ATP, 1.0 μl of 10×ligase buffer(Promega), and distilled water were added to a final volume of 9.5 μl.The DNA ends were ligated by incubation at room temperature for 2.5hours in presence of 2 units (0.5 μl) of T4 DNA ligase (Promega).

[0137] The E. coli JM 109 [recA1, supE44, endA1, hsdR17, gyrA96, relA1,thiΔ(lac-proAB) F′(traD36, proAB⁻, lac1⁴, lacZΔM15)] competent cellswere transformed with the ligated DNA by electro-transformation, usingthe Bio-Rad Gene Pulser apparatus. The competent cells were producedaccording the procedure described above and 40 μl of cell suspensionwere added to 2.0 μl of ligation mix. After incubation on ice for oneminute, the mixture was transferred into a cold 0.2 cm electroporationcuvette and pulsed with a time constant of four to five millisecondswith a field strength of 12.5 kV/cm. Immediately thereafter 1.0 ml ofprewarmed SOC medium was added to the mixture and incubated at 37° C.for one hour in an orbital shaker. About 50 μl aliquots were plated onSOB (2% Bacto trypton, 0.5% Bacto yeast extract, 8.5 mM NaCl, 2.5 mMKC1) plates containing 100 μg/ml ampicillin, X-gal and IPTG. After anovernight incubation at 37° C., single white recombinant colonies werepicked up, grown in 5.0 ml of SOB medium with 50 μg/ml of ampicillin andstored at −70° C. in 15% glycerol. Prior to the long term storage,further confirmation of the recombinants was ascertained by BamH I andEcoR I restriction digestion and agarose gel electrophoresis of miniprepDNA from +ve clones.

[0138] Expression of Recombinant Proteins in pRSET-C:

[0139] Each recombinant protein has different characteristics which canaffect optimal expression parameters. To overcome this situation a pilotexpression experiment was performed to determine the kinetics ofinduction for the 50 kD and 85 kD antigen genes. Briefly 2.0 ml of SOBmedia with 50 μg/ml of ampicillin were inoculated with a single whiterecombinant E. coli colony. The cells were grown at 37° C. overnight inan orbital shaker. The next day 50 ml of SOB media with 50 μg/mlampicillin was inoculated with 0.2 ml of the overnight culture and grownat 37° C. with vigorous shaking to an OD₆₀₀=0.3. An one ml aliquot ofthe culture was removed at this time point and centrifuged to pellet thecells. This was considered as the time zero sample and was frozen at−20° C. IPTG was added to the remaining culture to a final concentrationof 1.0 mM and the cells were grown in presence of IPTG for an additionalhour. After this time period the culture was inoculated with M13/T7phage (Invitrogen) at an optimal ratio of 5 pfu/cell. The infection wasallowed to proceed for another five hours at 37° C. and an one mlaliquot of culture was removed every hour. Each sample was centrifugedand both the supernatant and cell pellet was stored as before.

[0140] After all the samples were collected, the each pellet wasresuspended in 100 μl of 20 mM phosphate buffer (pH 7.0) and frozen inliquid nitrogen. The frozen samples were thawed again in a 42° C. waterbath and this freeze/thaw cycle was repeated an additional three times.Finally the freeze/thaw pellets were centrifuged at 14000×g for 10minutes in a refrigerated microcentrifuge and the supernatants with thesoluble protein fractions were transferred to a fresh tube. The pelletswith the insoluble protein fractions were also collected and resuspendedin 100 μl of Laemmli buffer. The supernatants were also mixed with equalvolumes of Laemmli buffer. Twenty μl of each sample (fractions of bothsupernatants and pellets) was electrophoresed separatly on a 10% SDSpolyacrylamide gel, following the identical procedures as describedabove. The gels were stained with Coomassie Blue and the bands werecompared for increasing intensity in the expected size range of the 50kD and 85 kD antigens to determine the optimal time point of maximumexpression.

[0141] The large scale extraction and purification of the recombinantproteins were accomplished under denatuing condition. To do this, 50 mlculture of the selected bacteria expressing the recombinant proteinswere harvested at the optimal time point of maximum expression. Thecells were pelleted by centrifugation at 5,000 rpm for five minutes in aSorvall SS-34 rotor and the pellets were resuspended in 10 ml ofguanidine lysis buffer (6 M guanidine-HCl, 20 mM NaPO₄, 500 mM NaCl).The temperature of the buffer was preadjusted to 37° C. for quick lysisof the cells, but to assure that complete lysis was obtained, the cellswere rocked at room temperature for additional 10 minutes. To shear theDNA and RNA, the cell lysates were sonicated on ice with three fiveseconds pulses at a high intensity setting. After the sonication, theinsoluble debris were removed from the sheared lysates by centrfugationat 3,000×g for 15 minutes and the clear lysates were stored at −20° C.for further purification with ProBond™ resin columns (Invitrogen).

[0142] The recombinant proteins expressed in the pRSET-C vectorcontained six tandem histidine residues in the amino terminal of thepeptides, which had a high affinity for ProBond™ resin. To bind therecombinant proteins in the columns, the resins of the columns wereresuspended with 5.0 ml of guanidine lysate of the expressed proteinsand rocked on an orbital shaker for 10 minutes at room temperature. Theresins were settled by gravity and supernatants were removed carefully.This step was repeated again with another 5.0 ml fresh aliquot of thelysates. After binding the proteins with the resins, the columns werewashed twice with denaturing binding buffer (8M urea, 20 mM NaPO₄, 500mM NaCl, pH 7.8), twice with denaturing wash buffer (8M urea, 20 mMNaPO₄, 500 mM NaCl, pH 6.0) and twice with the same denaturing washbuffer at pH 5.3. The washings were accomplished by simply resuspendedthe resins with 4.0 ml of each buffer for two minutes and thenseparating the resins from the supernatants by gravity. Finally thewashed columns were clamped in a vertical position and the cap wassnapped off on the lower end. The proteins were eluted from the columnsby applying 5.0 ml of denaturing elution buffer (8M urea, 20 mM NaPO₄,500 mM NaCl, pH 4.0). The elutes were collected and dialyzed against 10mM Tris, pH 8.0, 0.1% Triton X-100 overnight at 4° C. to remove urea,and then analyzed by Western blotting to confirm the specificity of theexpressed proteins.

[0143] Immunoblot Analysis of Ehrlichia risticii Component Antigens of25D and 90-12 Strains:

[0144] The antigenic composition profile of the variant (90-12) strainby Western blotting differed considerably from that of the 25D strain.Previous analysis of the Renografin purified standard (25D strain)indicated the presence of 18 component antigens of which nine (withmolecular weights of 110, 70, 68, 55, 51, 50, 33, 28, and 22 kD) weremajor antigens. I¹²⁵ surface labeling determined that the above antigenswere apparent surface antigens. Further analysis by Western blottingwith horse, rabbit and mouse antisera confirmed them as major antigens.Though several of these major antigens, namely the 68, 55, 49, and 28kD, proteins were similar in both strains, the main differences betweenthem were as follows: (i) The 110 and 70 kD antigens were present onlyin the 25D strain and they did not react with the 90-12 strain antisera.(ii) The 85 kD antigen was present only in the 90-12 strain, but itreacted with the 25D strain antisera. (iii) The 50 kD antigen waspresent only in the 25D strain and cross reacted with 90-12 strainantisera. (iv) The 55 and 51 kD antigen bands in the 25D strain werewell separated, whereas in the 90-12 strain they were close together asa 55/51 kD band. (v) The 33 kD antigen band of eaclystrainshowedcomparatively less color intensity, with the heterologous antiseras ascompared to the homologous antisera.

[0145] The Recombinant Antigens and Their Identity:

[0146] The recombinant clones expressing the partial or complete antigengenes were identified from two different genomic library of E. risticiistrains. A λ-gt11 recombinant library was constructed with 25D straingenomic DNA and a λ-ZAP recombinant library was constructed with 90-12strain genotnic DA.

[0147] A-gt11 Recombinants:

[0148] The recombinant clones expressing the 50 kD and 70 kD antigengenes of 25D strain was produced previously in λ-gt11 bacteriophage. The70 kD recombinant was obtained from a library generated by using thepartial Hpa II digest of E. risticii DNA. The 50 kD recombinant wasobtained from the library generated from E. risticii DNA subjected to acomplete double digestion with Hpa II and HinP I. After identificationof the recombinant antigens by the corresponding clone-specificantibodies, further analysis was conducted on 50 kD antigen gene as apart of this study.

[0149] λ-ZAP Recombinants:

[0150] The genomic expression library in λ-ZAP was Generated with Sau3AI digested E. risticii DNA. The Sau3A I digested fragments ranged insize from about 400 bp to 2 kb. The efficiency of production of λ-ZAPrecombinants was about 10⁷ pfu/μg of λ-ZAP DNA, of which about 8% werenon-recombinants. The number of antibody reactive recombinants was about10 to 14 per 10⁴ pfu. A total of 170 clones reactive with the 90-12strain antisera were picked up for further analysis. Clone-specificartiaen selected antibodies from these clones were prepared and reactedwith strips of transblotted 90-12 strain antigens. A comparison of theWestetn blots of these clone specific antigen selected antibodies withpolyclonal 90-12 antisera resulted in the identification of recombinantsexpressing the 85, 68, 55, 49, 33, 28, and 22 kD antigens. The 51 kDrecombinant clone was identified separately from an EcoR I library of90-12 spain generated in λ-ZAP system. After identification ofrecombinant antigens from this λ-ZAP library, further study wasconducted on the 85 kD antigen.

[0151] The Expression Characteristics and Cross Reactivity of 50 Kd and85 kD Recombinant Antigens:

[0152] A single recombinant clone expressing the 50 kD recombinantantigen was identified from the λ-gt 11 library of the 25D strain,whereas two different recombinant clones expressing the 85 kDrecombinant antigen were isolated from λ-ZAP library of the 90-12strain. Among these two recombinant antigens, the 50 kD antigen was anonfusion protein, expressed independently of the IPTG induction.Further analysis also revealed that the molecular mass of 50 kDrecombinant was identical to that of its native counterpart, indicatingexpression of the complete protein. Both 85 kD recombinant clonesexpressed a partial 85 kD antigen with β-galactosidase fusion. Theymigrated in the gel in conjunction with β-galactosidase, and theirexpression was dependent on IPTG induction.

[0153] It was discussed above that 85 kD and 50 kD antigens were notpresent in the 25D and 90-12 strains respectively, but these twoproteins were cross reacted with each other's strain specific antisera.Further analysis by Western blot also revealed that the recombinantclone expressing only the 50 kD antigen were cross reactive withantisera raised in mice specifically against the 90-12 strain and thesame way vice-versa with 85 kD recombinant clones. These observationsclearly indicated that the 50 kD and 85 kD antigens had theircorresponding cross-reactive counter part present in both strains. As anattempt to identify these corresponding crossreactive counter parts ofthe two proteins in each strain, a Western immunoblot was performed withthe 50 kD and 85 kD clone specific antibodies. It was observed that the50 kD clone specific antibody cross reacted with the 85 kD antigen ofthe 90-12 strain and the 85 kD clone specific antibody cross reactedwith the 50 kD antigen of the 25D strain. These results indicated thepresence of common cross-reactive epitopes in two different molecularweight proteins which were very strain specific and distinguishableserologically. These two homologous proteins were designated asstrain-specific antigens (SSA).

[0154] Nucleotide Sequence of 50 kD and 85 kD Recombinant Clones:

[0155] The complete sequence of the 50 kD antigen gene reading frame wasobtained from a single clone identified in the λ-gt11 library of the 25Dstrain where as a partial reading frame of the 85 kD antigen gene wasobtained from two separate overlapping clones identified in λ-ZAPlibrary of the 90-12 strain. The remaining sequence at the 5′ terminusof this gene was obtained later from a PCR amplified segment of the90-12 genomic DNA. Both the 50 kD and 85 kD insert pieces were subclonedseveral times to obtain nucleotide sequence information and identify thepossible open reading frame of both genes.

[0156] Subclones of 50 kD and 85 kD Recombinants:

[0157] EcoR I restriction digestion of the 50 kD λ-gt11 recombinantphage DNA, generated a 3.9 kb insert DNA fragment which was cloned inpBluscript SK(+) phagemid for restriction mapping. Fifteen restrictionenzymes (6 base-cutters) were used to determine the presence ofrestriction sites in the insert DNA of the above pBluscript SK(+)subclone. The Hind III digestion of the recombinant pBluscript SK(+) DNAproduced three DNA fragments of 3.5 kb, 2.2 kb and 1135 bp. The 3.5 kbDNA fragment was a plasmid-insert DNA piece, where 565 bp was an insertpart and the rest of it was pBluscript SK(+) phagemid. This specificfragment was re-circularized to form a pB50-6.1 subclone. The 2.2 kb and1135 bp insert fragments were subcloned separately in pBluscript SK(−)phagemid and they were designated as pB50-6.2 and pB50-6.3 respectively.It was difficult to select a primer for downstream sequencing of thepB50-6.2 recombinant clone, due to the presence of direct repeats in themiddle of the insert. In order to overcome this situation an internalsegment of 826 bp was PCR amplified by using two unique primers: 50-A(^(5′) ATA CTA AAA AGC ATA CTC ^(3′)) and 50-B (5′ TTC TAC AAG CCC TTTAAA ^(3′)). The amplified product was cloned in pCR™ vector anddesignated as pCR50-6.2.1. The insert piece of the pCR50-6.2.1recombinant clone was then easily sequenced by using the universalprimers of the vector. The presence of direct repeat motifs in thepB50-6.3 recombinant clone produced the same problem as described aboveand thus the insert piece of this clone was further subcloned in smallerfragments to exploit the advantage of the universal primer sequences forthe vector. For this purpose the restriction digestion was performedwith Pst I and the generated fragments were cloned separately inpBluscript SK(−) phagemids. Subclones were designated as pB50-6.3.1 andpB50-6.3.2.

[0158] The two in vivo excised phagemid clones partially expressing the85 kD antigen gene were designated as pB85-11 and pB85-17. The insertsize of these two clones were 4.5 kB and 1.1 kb respectively. These twoclones had 58% overlapping regions with each other and they togethercovered 84% of the 85 kD gene sequence. The remaining unknown 16% of the5′ region of the gene was separately cloned by PCR from 90-12 genomicDNA, using primers 50-C (^(5′) GAA TGT TCA GCT TTC CGG ^(3′)) and 50-D(^(5′) AGC TGT ATC GTT CGT GAG ^(3′)). The 1.5 kb amplified product wascloned in pCR™ II vector and designated as pCR85-3. The 3′ region of thegene was covered by the pB85-11 recombinant clone. The presence of toomany direct repeats in this region made the selection of sequencingprimers extremely difficult. To overcome this situation the insertsegment of this clone was further subcloned in smaller fragments toexploit the advantage of the universal primer sequences for the vector.For this purpose two primers, 85-E (^(5′) GTA TAC TTA CAG ATA GCA C^(3′)) and 50-E (^(5′) GCC GAC AGT ATC ATT AAA C ^(3′)), were used toamplify a 876 bp segment, using pB85-11 recombinant DNA as a template.The segment was cloned separately in a pCR™ II vector and designated aspCR85-11.1. The insert piece of pCR85-11.1 was restriction digested withHind III enzyme and as a result of this, two DNA fragments of 4.3 Kb and443 bp were produced. The 4.3 Kb fragment consisted of 495 bp insertpiece and the rest of it (3.8 kb) was the plasmid vector part. Thisspecific fragment was re-circularized to form the pCR85-11.1.1 subclone.The 441 bp fragment consisted of a 383 bp insert piece and a 60 bpplasmid piece. The 441 bp fragment was subcloned at the Hind III site ofthe pBluscript SK(−) phagemid and designated as pB85-11.1.2. Therecombinant DNA of pCRS5- 11.1.1 was double digested with Hind III andEcoR I. The generated fragments were purified from the agar gel by theGene clean technique and were further restriction digested with Sau3A Ienzyme, The Sau3A I digestion generated two fragments of 317 bp and 247bp. These fragments had a 9 bp and a 60 bp of plasmid sequence,respectively. These two pieces were separately subcloned at BarnH I-EcoRI and BamH I -Hind III sires of pbluscript SK(−) phagemid. They weredesignated as pBS5-11.1.1.1 and pB85-11.1.1.2 respectively.

[0159] Sequence of 50 kD and 85 kD Recombinant Clones:

[0160] Two vector primers from the opposite direction were used toreveal the complete sequence of a 565 bp insert fragnent of the pB50-6.1recombinant. Sequence analysis of this region did not indicate thepresence of any possible reading frames for the 50 kD antigen gene. Thecomposite sequence analysis of the pB50-6.2 and pCR50-6.2.1 recombinantsindicated a possible reading frame for the 50 kD antigen gene present inthe 2.2 kb fragment of pB50-6.2. The first methionine was located at the848 bp downstream of the 5′ end of this fragment, and the reading framewas continued all the way to it's 3′ end. Further sequence analyses ofthe pB-50-6.3.1 and pB50-6.3.2 recombinants revealed the completesequence profile of the 50 kD antigen gene.

[0161] Sequence analysis of the pB85-17 recombinant clone of the 90-12strain helped to identify the presence of an 1155 bp uninterruptedreading frarne of the 83 kD antigen gene. However the fragment did notcontain the 5′ or 3′ end of the gene. Further analysis of this clonerevealed a partial sequence homology with the the 50 kD gene of the 25Dstrain which helped in the selection of the two primers 50-C and 50-Dfor amplifing the 5′ end of the gene. The sequence analysis of thecloned, amplified product (recombinant pCR85-3) revealed the 5′ end ofthe gene. Analyses of the reading frames for subclone pCR85-11.1,pCR85-11.1.1, pCR-11.1.2 pB85-11.1.1.1 add pB85-11.1.1.2 exposed thecomplete 3′ end sequence information of this gene.

[0162] Genomic Localization of 50 kD and 85 kD Strain Specific AntigenHomologues:

[0163] The presence of a variable number of tandem repears in the ORFsof both the 50 kD and 85 kD antigen genes, increases the possibilitythat these genes might be residing in a multigene family category. Thusthere may be more than one copy of these genes, with other variablenumbers of repeats, present somewhere in the chromosome. To confirmthis, a specific probe of 1.5 kb was generated by PCR from the 90-12genomic DINA following the procedure described above. Thae primers foramplification were selected in such a way that the amplified productcontained a common 697 bp upstream and 180 bp downstream regions fromthe first methionine of the oth 50 kD and 85 kD antigen genes. As acontrol, the insert segment of two ocher recombinant clones expressingthe 55 kD and 51 kD antigen genes of the 90-12 strain were used as aprobe.

[0164] The α³²P labeled probes were hybridized to the E. risticiigenomic DNA of the 25D and 90-12 strains. The genomic DNA of bothstrains were digested with EcoR I and HinD III. Since Sau3A I was thesingle restriction enzyme used to obtain λ-ZAP recombinants, E. risticiiDNA of both strains, digested with Sau3A I, were also used for theidentification of homologous genomic DNA fragments in theserecombinants.

[0165] The probes made with the inserts of the 55 kD and 51 kDrecombinant clones of the 90-12 strain hybridized with the same-sizefragments in each of the three restriction enzyme digests of bothstrains.

[0166] Molecular Structure of Ehrlichia risticii SSA Homologues:

[0167] The molecular structure of E. risticii of the SSA homologues (50kD and 85 kD) antigen genes were revealed by analyzing the completenucleotide and amino acid sequences of these two proteins. The completenucleotide sequences of the genes were constructed from the sequences ofindividual clones and their subclones. Due to the presence of severaldirect repeats in these genes, the sequences obtained from theoverlapping and adjoining clones and their subclone were furtherconfirmed by amplification and sequencing of those areas directly fromthe genomic DNA of their respective strains.

[0168] Nucleotide Sequence Analysis:

[0169] A total of 2632 bp (25D strain) and 3357 bp (90-12 strain) weresequenced in the cloned E. risticii DNAs. The nucleotide sequence of thecloned 25D strain consisted of 869 bp of 5′ noncoding region, 1617 bp ofthe ORF, and 146 bp of a 3′ noncoding region. The nucleotide sequence ofthe cloned 90-12 strain consisted of 696 bp of a 5′ noncoding region,2547 bp of the ORF, and 114 bp of a 3′ noncoding region. The basecompositions of the sequenced DNAs showed high A+T contents (70%),especially in the 5′ and 3′ noncoding regions (71-80%). This reflects ahigh A+T-rich genomic DNA in Ehrlichia.

[0170] Structure of the 50 kD Antigen Gene:

[0171] The nucleotide sequence of the 50 kD antigen gene ORY and 5′ and3′ flanking regions were determined and the amino acid sequence wasdeduced and depicted (FIG. 3, SEQ ID NO: 3 and 4). An ATG Eslation startsite at base pair position 175 and a TAA termination site at base pairposition 1792 completed an ORF of 1617 nucleotides encoding 539 aminoacids. The deduced sequence of the 50 kD antigen has a calculatedmolecular mass of 59.829 kD, which is in reasonably close agreement tothe size originally observed on SDS-PAGE. The possible transcriptioninitiation site and upstream control region are indicated in FIG. 3. Theupstream control region contained nearly perfect −10 and −35 consensusprokaryotic promoter sequences.

[0172] The ORF of the 50 kD antigen gene continued uninterrupted atleast 66 bp upstream of the proposed ATG translation start site. This 5′region had no ATG codons present which could potentiate anothertranslation initiation site. The further 5′ upstream region of this genehad two ATG codons which may be considered as translation initiationsites, but there were two distinct stop signal within 50 basesdownstream of these two ATGs. Also, the recombinant in the expressionvector produced a full-length product, while lacking the region 5′ ofthe proposed ATG. These two pieces of evidence nullified the possibilityof these two ATGs as a translation initiator. The space between the −35and −10 regions was 17 bp, which is consistent with the optimal spacing(17±1) for prokarvotic promoters. The sequence GAAAAA at 7 bp upstreamfrom the start codon was identified as a potential ribosome-binding sitefor m-RNA translation.

[0173] The non-coding region downstsream of the translation terminationsite was a 143 bp stretch containing inverted repeats bordered by athymine rich region, resembling prokavotic rho-independent transcriptionterminators. These features are denoted in FIG. 3.

[0174] Structure of the 85 kD Antigen Gene:

[0175] The nucleotide sequence of the 85 kD anttger gene ORF and 5′ and3′ flanking regions were determined and the amino acid sequence wasdeduced (FIG. 2, SEQ ID NO: 5 and 6). An ATC translation start site atbase pair position 175 and a TAA termination site at base pair position2722 completed an ORF of 2547 nucleotides encoding 849 amino acids. Thededuced sequence of the 85 kD antigen has a calculalated molecular massof 94.333 kD, which is in reasonably close agreement to the sizeoriginally observed on SDS-PAGE. The possible transcription initiationsite and upstream control region are indicated in FIG. 2. The upstreamcontrol region, the translation start site, and first 178 bp after thefirst ATG were almost identical with the 50 kD antigen gene sequence.The ORE of 85 kD antigen gene continued uninterrupted at least 66 bpupstream of the proposed ATG translation start site. Like the 50 kDantigen gene sequence, this 5′ region had no ATG codons present whichcould potentiate another translation initiation site, and it did notaffect the full-length expression of the 85 kD antigen, as therecombinant expression vector produced a full-length product whilelacking the region 5′ of the proposed ATG. The proposed ribosome-bindingsite GAAAAA was present 7 bp upstream from the start codon.

[0176] The non-coding region downstream of the translation terminationsite was a 112 bp stretch containing inverted repeats bordered by athymine rich region, resembling prokaryotic rho-independenttranscription terminators. These features are denoted in FIG. 2.

[0177] Repeat Motifs and Their Nature in 50 kD and 85 kD SSA Homologues:

[0178] The DNA sequence analyses of the 50 kD and 85 kD antigen genesrevealed the presence of several direct repeats in both genes. Thefrequency of these repeats were more in middle of the genes and many ofthese repeats were identical in both genes. All these identical repeatscoded for same amino acids but the position and the frequency ofrepetition were quite different in both genes. TABLE 1 Repeat locationsand sequences along the 50 kD antigen gene. Analyses were conducted onthe 11-base repeats. There were 12 different types of 11-base repeatspresent in the complete sequence of the gene. A total of thirty-four11-base direct repeats were identified in the gene. Type of RepeatsRepeat Sequence Repeated from Base I AAAGAAATACT 957, 1434, 777, 1287,1353, 648. II GAAATACTCAC 807, 1356, 651, 1290, 1383. III AAATTTAAAGA978, 1242, 852, 1110, 915. IV CTAAAAGAGAT 510, 1017, 891, 1149. VAAAGACATACT 501, 1071. VI TTTAAAGAGCT 342, 1113. VII ATTTTTTATAA 75,119. VII AACTTTAAAGG 408, 1179. IX AAGTTTAAAGA 339, 1584. X TACTCACTAAT457, 1504. XI AGTTTAAAAAA 669, 1309. XII ATAAGTTTAAA 237, 288

[0179] Repeats in the 50 kD Gene:

[0180] There were a total of 97 repeats present in the 50 kD antigengene sequence. These repeats were not totally identical in their lengthsand sequences. Tney were first categorized according to their lengthsand the, under the same lengths they were grouped according to theirsequence profiles. The minimum length of these repeats was 10 bases,whereas the maximum length was 38 bases. The result of these Analysesare represented in Table 1.

[0181] Repeats in the 85 kD Gene:

[0182] The structures of repeats in the 85 kD antigen gene were almostidentical to the 50 kD antigen gene. There are a total of 356 repeatspresent in this gene sequence. As for the 50 kD antigen gene, theserepeats were categorized according to their lengths and then, under thesame length, they were grouped according to their sequence profiles. Themaximum and minimum lengths of these repeats were 55 and 10 basesrespectively. As with the 50 kD antigen gene the 11-mer repeats werealso abundant in the complete sequence of the 85 kD antigen gene, andthey also were further analyzed for their specific positions in thesequence. The results of these analyses are presented in Table 2. TABLE2 Repeat locations and sequences along the 85 kD antigen gene. Analysewere conducted on the 11-base repeats. There were 20 different types of11 base repeats in the complete sequence of the gene. A total onehundred and one 11-base direct repeats were identified in the gene. Typeof Repeats Repeat Sequence Repeated From Base. I ATACTTACAGA 652, 1963,1300, 901, 832, 385, 2260, 1729, 316. II AAATTTAAAGA 1984, 2116, 1852,1390, 1252, 853, 784, 1915. III CTAAAAGAGAT 1891, 2023, 1429, 892, 760,376, 1228. IV AAAGAAATACT 1696, 1567, 1165, 2227, 1030, 2161, 646. VTACTTACAGAT 1064, 1964, 834, 902, 1301, 1730, 2261. VI AAAGACATACT 310,1945, 1351, 1282, 883, 814, 367. VII ACAGCTAAAGA 2275, 2302, 1771, 1744,2302, 1159. VIII TTTAAAGAACT 1393, 2515, 2185, 856, 1323, 339. IXGAAATACTTAC 2164, 2257, 641, 1168, 1726. X AGCACTGGTAA 1975, 2005, 1381,844, 1312. XI GATAAATTTAA 1912, 2380, 781, 1249, 1849. XII CTTATAGAAAG934, 1333, 865, 349, 550. XII GAAATACTCAC 676, 2230, 1699, 1570, 1033.XIV ACCGGTAACTT 532, 916, 1433, 2047. XV ATGCAACAAAA 2204, 2621, 1007.XVI GCTAAAGAAGT 1189, 2278, 1747. XVII CTTACAGATAA 904, 2035, 1441.XVIII GCAATAACTGG 733, 1864. XIX ATGGTAAGGAC 494, 746. XX ACTTATAGAAG417, 1401.

[0183] Analysis of Deduced Amino Acid Sequences of SSA Homologues:

[0184] The amino acid sequence analyses of the 50 kD and 85 kD antigengenes indicated a considerable homology between these two SSAhomologues. That the identical repeats of these two genes code for thesame amino acids, indirectly indicates a conserved region between thesetwo genes. From a comparison of the 32 amino acid sequences encoded inthe N-terminal ends of the 50 kD and 85 kD antigens an almost identicalsignal sequence was identified for both proteins. Only one substitutionof leucine for isoleucine occurred at residue 26 of the amino acidsequence in the 90-12 strain. These signal peptides for both strainsconsist of a polar region and a hydrophobic core, of which the samecharacteristics are seen in the signal peptides of other prokaryoticcells. The hydrophobic core region is extended from the 16th to 28thresidues in the signal sequence. The predicted processing site of thesignal peptide is at the bond between the 31st and 32nd amino acids,with isoleucine as the N-terminal amino acid of the mature SSA in bothcases.

[0185] Amino acid sequence comparison of the SSAs of these two antigenicvariants is presented in FIG. 5 (SEQ ID NO: 4 and 6). In these analyses,substitution or the addition of one or several contiguous amino acidresidues were identified throughout the molecules, but the significanthomology in amino acid sequence of the 50 kD and 85 kD antigen was verypronounced in certain regions of tre two molecules. These specific areaswere designated as ID (identical domain) I-VIII in FIG. 5. The mostinteresting feature of these IDs was the unique distribution of domainsin the linear amuno acid sequence of individual antigens. The domainswere positioned one after another (ID I to ID VIII) in the 50 kDantigen, whereas the positioning of the same domains was totallydifferent in the 85 kD antigen. In these ID regions, the similarities inthe amino acid sequences between these two individual strains vary frommore than 94% to less than 79%.

[0186] ID I is the largest identical domain, consisting of 129 aminoacids. Here the amino acid sequence of the 50 kD and 85 kD antigens werevery similar, and estimated homology is 89.15% (87.08% in nucteotidesequence) with 14 amino acid conversions. The position of thisparticular domain was the same in primary structures of both theantigens. This domain contained ache signal sequence resion of the theSSA homoloagues.

[0187] ID II consists of 51 amino acids. When comparing SSA homotogues,this particular domain is found further downstream in the 85 kD antigen.Here the estimated homology was 88.24% (89.54% in nucleotide sequence)with six amino acid conversions in between the 50 kD and 85 kD antigens.

[0188] ID III consists of 42 amino acids. The estimated homology is92.85% (92.06% in nucleotide sequence) with 3 amino acid conversions.This particular domain is also found further downstream in the 85 kDantigen as compared to the 50 kD antigen.

[0189] ID IV consists of 21 amino acids. Here the estimated homology inamino acid sequence is 90.48% (85.71% in nucleotide sequence) with 2amino acid conversions. With respect to the 50 kD antigen thisparticular domain is found fwurter upstream in the 85 kD antigen.

[0190] ID V consisted of 39 amino acids. Among all the domains, thisarea had the minimum homology of 79.49% (80.34% in nucleotide sequence)in SSA homologues. In the 85 kD antigen this domain is found furtherupstream as compared to the 50 kD antigen.

[0191] The ID VI domain region has the maximum homology of 94.55%(93.82% in nucleotide sequence) between the two antigens. Similarly, theID VII and ID VIII domains possess the high homologoy. ID VII has 92.11%homology (85.08% in nucleotide sequence) and ID VIII has 94.12% homology(96.73% in nucleotide sequence) in their respective areas of the SSAhomologues.

[0192] After comparing the position of all the identical domains in SSAhomologues it is clear that six domains out of eight are changed withrespect to their positions in these antigens. In the 85 kD antigen thedomains are farer apart from each other as compared to the 50 kDantigen, and these gaps are filled with new sequences. Theseobservations indirectly indicate the generation or more new anddiffereit domains in the 85 kD antigen.

[0193] Hydropathy Analysis of SSA Homologues:

[0194] Hydropathy analysis showed that the SSAs of both strains havealternative hydrophilic and hydrophobic motifs which are characteristicof transmnembrane proteins. The hydropathy plot of the 50 kD antigenrevealed four major hydrophobic stretches which are sufficient in lengthand hydrophobicity to serve as transmembrane domains. The largesthydrophobic stretch belorngs to the identical domain I, and forms thehydrophobic core region of the predicted signal peptide. The other threehydrophobic stretches are clustered in last 60 amino acids of theC-terminus of the protein. Hydropathy analysis of the 85 kD antigenindicats the presence of at least eight major hydrophobic regions. Anyone of these regions can act as a transmembrane domain. Like the the 50kD antigen, this antigen also possesses the largest hydrophobic regionin its identical domain I, and other three hydrophobic regions in thelast 60 amino acids of the C-terminus. The other four major hydrophobicregions are distributed between residue 200 and residue 410 in thesequence. Hydrophilicity indices for both antigens indicated thepresence of many outer membrane domains which may be exposed on theouter surface of the organism or the inner cytoplasomic side of themembrane.

[0195] Epitope Analysis of SSA Homologues:

[0196] Locating the possible antigeric determinants by analyzing proteinamino acid sequences in order to find the point of greatest locaihydrophilicity, is a corrnon technique nowadays. This was accomplishedby assigning each atino acid a numerical value (hydroplulicity value)and then repetitively averaging those values along the peptide chain.The point of highest local average hydrophiliciry was invariably locatedin, or immnediately adjacent to, an antigenic detemunan or epitope.Using this technique combined with analysis of the flexibility ofproteins, the possible antigenic determinants of the 50 kD and 85 kDantigens were determined. Analysis of the comparative position of theseepitopes in the common domains of the 50 kD and 85 kD antigens wascritical to the evaluate the presence of possible cross-reactive andstrain specific antigenic determinants in the 25D and 90-12 strains.

[0197] In order to compare the structurai as well as antigenic aspectsof the SSA homologues, Chou-Fasman predictions of the secondarystructure of both the 50 kD and 85 kD complete antigens were plotted.None of these plots were identical to each other. Those regionspredicted to have a high likelihood of antigenicity were also determinedby the algorithm of Jameson and Wolf. Several regiorns of high antigenicindices appeared to be conserved in both the antigens, although theirpositions and orientations in the secondary structure are quitedifferent. Analysis of antigenicity of the 50 kD indicated nine majorareas with high antigenic indices (residues 76-80, 118-122, 274-278,332-336, 362-366, 478-482, 508-512, 518-522, and 528-532). Among thesenine major areas, the first two belong to ID-I; the 3rd one belonged toID-IV; the 4the and 5the, to ID-VI; and the last four to an unique aminoacid sequence region of the 50 kD antigen which had no homology with the85 kD sequence. Analysis of antigenicity of the 85 kD antigen indicatednine major areas with high antigenic indices (residues 76-80, 108-112,118-122, 212-216, 246-220, 426-430, 590-595, 622-627, 844-848). Amongthose nine major areas, the first two belonged to ID-I, and th 3rd, 4theand 5the to ID-IV, ID-V and ID-VI respectively. The last three belongedto an unique amino acid sequence region of the 85 kD antigen which hadno homology Nith the 50 kD antigen sequence. Several regions of highantigenic index in both antigens appeared to be conserved (residues76-80, 118-122, 274-278, 332-336 in the 50 kD antigen and 76-80,108-112, 118-122, 212-216, 426-430 in the 85 kD antigen). A highantigenic index region in the 85 kD antigen belonged to ID-V, where asthe ID-V in the 50 kD antigen does not possess such type of highantigenic index region. This type of variation in this region of boththe 50 kD and 85 kD antigens was predicted because the homology betweenthe ID-V's in SSA homologues was minimum (79.49%) when compared to theother identical domains of these two antigens.

[0198] Recombinant Antigens and Their Characteristics:

[0199] The complete ORF of the 50 kD and 85 kD antigens were constructedby PCR and cloned in pRSET-C expression vector. The correct ORF of thegenes were confirmed by cloning and sequencing the PCR amplified productseparately in pBluescript SK(−) pnagemids prior to expression.

[0200] SK(−) Recombinant Clones of the 50 kD and 85 kD Antigens:

[0201] The mnotecular size of the PCR generated fragments whichcontained the full length genes of the 50 kD and 85 kD antigens were1.61 kb and 2.54 kb respectively. They were cloned separately in SK(−)phagemids. The BamH I-EcoR I restriction digestions of the recombinantphagemids generated right size inserts, which were expected from thesequence information for these genes. Sequence analyses of theserecombinant inserts confirmed the correct amplification of the SSA genesdirectly from their respective strains.

[0202] pRSET-C Recombinant Clones of the 50 kD and 85 kD Antigens:

[0203] Total 18 positive pRSET-C recombinant clones of the 50 kD and 85kD anticen genes (nine for each gene) were separately analyzed byrestriction digestions to confirm the proper transfer of inserts fromSK(−) phagernids to pRSET-C expression vectors. All nine positive clonesfrom the 50 kD recombinants were successfully transferred in expressionvectors, whereas in the 85 kD group only four of the clones weresuccessfully recombined with the expression vectors. Finally, thecomplete 50 kD and 85 kD antigens were expressed in the pRSET-C systems.Coomassie Blue staining of expressed proteins indicated that maximumexpression was achieved four to five hours after the IPTG induction.

[0204] Western Blot Analysis of the 50 kD and 85 kD Expressed Proteins:

[0205] The identities of the expressed proteins were established to bethe 50 kD and 85 kD antigens by the reactivities of E. risticii (25D and90-12 strains) polyclonal antisera and the 85 kD clone specific antibodywith the 50 kD and 85 kD antigens of their respective strains andcorresponding expressed proteins. Both the 50 kD and 35 kD antiaensmigrated anomalously during electrophoresis and appeared to be 9.0 kDsmaller than the encoded sizes.

EXAMPLE 2 Isolation of Strain Specific Surface Antigen Gene of Ehrlichiaristicii ATCC Type Strain

[0206] Using the procedures outlined in Example 1, the gene encoding the50 kDa SSA from the ATCC type strain was isolated. The gene sequence andthe amino acid sequence encoded thereby is shown in FIG. 4 (SEQ ID NO: 7and 8).

EXAPLE 3 Challenge Experiments Summary

[0207] To study the role of major antigens of E. risticii in protectiveimmune response, we expressed the genes of the 55 kDa, 51 kDa and 85/50kDa-strain-specific antigens of the 90-12 85 kDa antigen and 25-D (50kDa antigen strains in Escherichia coli. Mfice immunized with thesepurified recombinant proteins of E. risticii developed strong andspecific humoral immune response. The recombinant 85 kDa antigen of the90-12 strain protected mice against challenge infection with both E.risticii strains, whereas its homologue from the 25-D strain, therecombinant 50 kDa antigen, protected mice against only the homologousstrain challenge, but not against the heterologous 90-12 strain. Serafrom mice immununized with the 85- or 50-kDa antigens did not inibit thereplication of cell-free Ehriichia in in vitro neutralization assays.Sera from normal mice and mice iimunized with other antigens causednon-specific neutralization of E. risticii. Immunoglobulin 0 from miceimmunized with the 51 kDa protein of the 90-12 strain caused partial invitro neutralization of both strains of E. risticii. These studiesdemonstrate that the 85/50-kDa-strain-specific antigen of E. risticii isinvolved in immunoprotection against PHF.

Results

[0208] The protective capabilities of the purified recombinant antigersof R. risticii were tested in mice. In a pilot experimrent, the 51 kDa,55 kDa, 85 kDa, and 51+85 kDa antigens of the 90-12 strain were used toimmunize the mice. Immunizations were performed by intrapenitonealinoculation of the respective antigen(s). The antibody response of miceto the recombinant antigens was deterrnined by IFA using MM cellsinfected with the 90-12 strain. The prechallenge serum antibody items ofthe different experimental groups are shown in FIG. 6. The antibodytiters varied from 1/40 to 1/640. The 85 kDa and 51+81 kDa groups ofmice contained higher titers compoared to the mice in the 51 kDa and 55kDa groups. After the challenge infection with the 90-12 strain, themice in 51, 85, 51+85 kDa, and the 90-12 organism groups did not showany clinical signs up to 21 days post-challenge. The 55 kDa and adjuvantgroups showed only mild clinical sips.

[0209] In a second experiment, the 50, 85, 51+85 kDa antigens of the90-12 strain, and the 51, 50, 51+50 kDa antigens of the 25-D strain wereincluded in the experimental groups. As positive controls, the mice wereimmunized with the purified organisms of the 90-12 and 25-5 D strains.The negative controls included the 55 kDa antigen, adjuvant, anduninoculated groups. At the time of the challenge infection, the serumantibody titers of these mice against the 90-12 strain (IFA titers) wereobtained (FIG. 7). After challenge infecting with the 90-12 strain, micein the 85 kDa and 51+85 kDa groups showed significant protection (FIG.8). In the 85 kDa immunized group, only two out of eight mice sufferedmild clinical signs for one and two days respectively. In the 51+85 kDaimmunized group one mouse suffered from the infection and it died on day12 post-challenge. The prechallenge serum antibody titer of this mousewas comparatively lower than the rest of the mice in that group. Mice inthe positive control groups were completely protected from theinfection. Mice in the negative control groups suffered from theclinical infection. The clinical signs of the mice immunized with eitherstrains 51 kDa antigen were less severe compared to those of thenegative control groups.

[0210] In a third experiment, the mice were challenge infected with the25-D strain. Even in the negative controls, the severiry of theinfection was less, thus confirtning the lower pathogenicity of the 25-DStrain. The 55 kDa immunized mice suffered mild clinical signs for onlytwo days. None of the experimental groups showed any clinical signs.

Discussion

[0211] The various challenge experiments described herein indicate thatthe recombinant strain-specific antigens, primarily the 85 kDa antigenof the 90-12 strain or the 90-12 strain itself can be used forimmunization purposes. Any variants of E. risticii that bind to theantibodies to the 85 kDa antigen of the 90-L2 strain may also be usedfor an attenuated bacterial vaccine At present, vaccine effectiveness ofexisting PHF vaccines is low, and it is believed that the presentinvention can provide a superior vaccine against PHF.

[0212] Also, the antigens disclosed herein can be utilized in diagnostictests or test kits to diagnose PHF in horses. In addition, the nature ofthe repeated sequences of SSA can be used to generate intragenic primersto obtain specific DNA amplification finger printing (DAF) todifferentiate various strains of E. risticii. The DNA amplificationfmger printing (DAF) of field E. risticii isolates are shown in FIG. 9.

[0213] The following references are incorporated herein by reference intheir entirety:

[0214] U.S. Provisional Application Serial No. 60/059,252, filed on Sep.18, 1997;

[0215] Biswas, Biswajit, Molecular basis of antigenic variation ofstrain-specific surface antigen gene of Ehriichia risticii anddevelopment of a multiplex PCR assay for differentiation of strains,Ph.D. Tresis, Univ. of Maryland, College Park, Md., USA SO (1996), 186pp. Avail.: Univ. Microfilms Int., Order No. DA9707569 From: Diss.Abstr. Int., B 1997, 57(10), 6067;

[0216] Vemulapalli, Ramesh, Miolecular analysis of differences betweentwo strains of Ehrichia risticii and identification of protectiveantigen, Ph.D. Thesis, Univ. of Maryland, College Park, Md., USA, SO(1996) 176 pp., Avail.: Univ. Microfilms Int., Order No. DA9707676,From: Diss. Abstr. Int., B 1997, 57(10), 6125;

[0217] Vemulapalli et al, Veterinary parisitology, 76, (1998), pp.189-202; and

[0218] Dutta et at, Jourral of Clinical Vicrob iology, February 1993,pp. 506-512.

[0219] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced othenvise than as specifically describedherein.

1 48 1 33 DNA Artificial Sequence Description of Artificial Sequencesynthetic primer 1 cataaaattt ctaagacgaa ggatccctat gtc 33 2 33 DNAArtificial Sequence Description of Artificial Sequence synthetic primer2 gagagaaagt tccccgtgtg aattctagct agg 33 3 2836 DNA Ehrlichia risticiiCDS (175)..(2721) 3 attggatcta aataatgtac actggaggtt cgtattttctattatgaaag ggatagaatg 60 ttaaatttta tgatttttta taataaaaat agatataaaatttagtagtt ttataaattt 120 ttcataacaa aggactatcc tccttgcata aaatttctaagacgaaaaat ccct atg 177 Met 1 tca aat gaa aca ctt ttg agc gta ctt tctgat gaa acg cac ttt gct 225 Ser Asn Glu Thr Leu Leu Ser Val Leu Ser AspGlu Thr His Phe Ala 5 10 15 aat cta gtt gat gaa ctt ctt ctc atc ttg gttaaa gac agt att ttc 273 Asn Leu Val Asp Glu Leu Leu Leu Ile Leu Val LysAsp Ser Ile Phe 20 25 30 act caa gta ata aaa ggc gag gga aag aca gaa ttaaaa gac ata ctt 321 Thr Gln Val Ile Lys Gly Glu Gly Lys Thr Glu Leu LysAsp Ile Leu 35 40 45 aca gac aac act ggt aag ttt aaa gaa ctt ata gaa agtgca ggt aaa 369 Thr Asp Asn Thr Gly Lys Phe Lys Glu Leu Ile Glu Ser AlaGly Lys 50 55 60 65 gac ata cta aaa gag ata ctt aca gac aat acc ggc aatttt aaa gga 417 Asp Ile Leu Lys Glu Ile Leu Thr Asp Asn Thr Gly Asn PheLys Gly 70 75 80 ctt ata gaa ggt aat ggt aag acg gag gca aaa gag gta cgcact aat 465 Leu Ile Glu Gly Asn Gly Lys Thr Glu Ala Lys Glu Val Arg ThrAsn 85 90 95 gaa aaa ttc aag gag ctt ttt gga agc aat ggt aag gac ata ctgaaa 513 Glu Lys Phe Lys Glu Leu Phe Gly Ser Asn Gly Lys Asp Ile Leu Lys100 105 110 gac att ctt act gat aac acc ggt aac ttt aaa ggc ctt ata gaaagt 561 Asp Ile Leu Thr Asp Asn Thr Gly Asn Phe Lys Gly Leu Ile Glu Ser115 120 125 gca gct aag ggt aag ctg aaa gat ctt ctt att gat gaa aaa tttcaa 609 Ala Ala Lys Gly Lys Leu Lys Asp Leu Leu Ile Asp Glu Lys Phe Gln130 135 140 145 aaa tta ttc gag gat gaa acg aaa gct ggt cgt gta aaa gaaata ctt 657 Lys Leu Phe Glu Asp Glu Thr Lys Ala Gly Arg Val Lys Glu IleLeu 150 155 160 aca gac agc aac gct aag gaa ata ctc aca aat gaa gta gcaaaa gag 705 Thr Asp Ser Asn Ala Lys Glu Ile Leu Thr Asn Glu Val Ala LysGlu 165 170 175 gta cta aaa tcc gat aaa ttc aag gag gca ata act ggc gatggt aag 753 Val Leu Lys Ser Asp Lys Phe Lys Glu Ala Ile Thr Gly Asp GlyLys 180 185 190 gac gca cta aaa gag ata ctt act tgt gat aaa ttt aaa gaggct gta 801 Asp Ala Leu Lys Glu Ile Leu Thr Cys Asp Lys Phe Lys Glu AlaVal 195 200 205 aca ggc aat ggt aaa gac ata cta aaa ggt ata ctt aca gatagc act 849 Thr Gly Asn Gly Lys Asp Ile Leu Lys Gly Ile Leu Thr Asp SerThr 210 215 220 225 ggt aaa ttt aaa gaa ctt ata gaa agt act agt aaa gacata cta aaa 897 Gly Lys Phe Lys Glu Leu Ile Glu Ser Thr Ser Lys Asp IleLeu Lys 230 235 240 gag ata ctt aca gat aat acc ggt aac ttt aaa ggc cttata gaa agc 945 Glu Ile Leu Thr Asp Asn Thr Gly Asn Phe Lys Gly Leu IleGlu Ser 245 250 255 act ggc aag gag aaa gta aaa gaa ctt ctt atc gat gggaag ttt aag 993 Thr Gly Lys Glu Lys Val Lys Glu Leu Leu Ile Asp Gly LysPhe Lys 260 265 270 gac ctg ttt act gat gca aca aaa gcc ggt tat gta aaagaa ata ctc 1041 Asp Leu Phe Thr Asp Ala Thr Lys Ala Gly Tyr Val Lys GluIle Leu 275 280 285 acg aac gat aca gct aag gaa gta ctt aca gat caa acagca aag gag 1089 Thr Asn Asp Thr Ala Lys Glu Val Leu Thr Asp Gln Thr AlaLys Glu 290 295 300 305 gtc cta aaa gat agt aca gct aaa gac ata tta aaggac aca aac gca 1137 Val Leu Lys Asp Ser Thr Ala Lys Asp Ile Leu Lys AspThr Asn Ala 310 315 320 gct gcg gta cta aaa aac agc aca gct aaa gaa atactt aca aac caa 1185 Ala Ala Val Leu Lys Asn Ser Thr Ala Lys Glu Ile LeuThr Asn Gln 325 330 335 acc gct aaa gaa gtg ctt aca gat ggt aca tcc aaagaa gta cta aaa 1233 Thr Ala Lys Glu Val Leu Thr Asp Gly Thr Ser Lys GluVal Leu Lys 340 345 350 gag ata ctt act tgt gat aaa ttt aaa gag gca gtaaca gga gat ggt 1281 Glu Ile Leu Thr Cys Asp Lys Phe Lys Glu Ala Val ThrGly Asp Gly 355 360 365 aaa gac ata cta aaa ggt ata ctt aca gat agc actggt aag ttt aaa 1329 Lys Asp Ile Leu Lys Gly Ile Leu Thr Asp Ser Thr GlyLys Phe Lys 370 375 380 385 gaa ctt ata gaa agt act ggt aaa gac ata ctgaaa gac att ctt aca 1377 Glu Leu Ile Glu Ser Thr Gly Lys Asp Ile Leu LysAsp Ile Leu Thr 390 395 400 gat agc act ggt aaa ttt aaa gaa ctt ata gaagta ctg gta aag aac 1425 Asp Ser Thr Gly Lys Phe Lys Glu Leu Ile Glu ValLeu Val Lys Asn 405 410 415 aag cta aaa gag att ctt aca gat aac acc ggtaac ttc aaa ggg ctt 1473 Lys Leu Lys Glu Ile Leu Thr Asp Asn Thr Gly AsnPhe Lys Gly Leu 420 425 430 gta gaa ggc gcc ggg aag gat gaa gca aaa gcagta ctt act gac gag 1521 Val Glu Gly Ala Gly Lys Asp Glu Ala Lys Ala ValLeu Thr Asp Glu 435 440 445 aaa ttt aaa ggc ttg ttt gat gac aaa aca atagct ggc tat gta aaa 1569 Lys Phe Lys Gly Leu Phe Asp Asp Lys Thr Ile AlaGly Tyr Val Lys 450 455 460 465 gaa ata ctc acc agc gag aag ttt aaa aaactg ttt gaa agt gca ggt 1617 Glu Ile Leu Thr Ser Glu Lys Phe Lys Lys LeuPhe Glu Ser Ala Gly 470 475 480 aag act aaa gta aaa gaa ctc ctc att gatgag aag ttt caa aaa tta 1665 Lys Thr Lys Val Lys Glu Leu Leu Ile Asp GluLys Phe Gln Lys Leu 485 490 495 ttt gag gat gac acg aaa gcc agt cat gtaaaa gaa ata ctc acg aac 1713 Phe Glu Asp Asp Thr Lys Ala Ser His Val LysGlu Ile Leu Thr Asn 500 505 510 gat aca gct aag gaa ata ctt aca gat caaaca gct aaa gaa gtc cta 1761 Asp Thr Ala Lys Glu Ile Leu Thr Asp Gln ThrAla Lys Glu Val Leu 515 520 525 aag gat agt aca gct aaa gag ata tta aaggac aca aac gca gct gcg 1809 Lys Asp Ser Thr Ala Lys Glu Ile Leu Lys AspThr Asn Ala Ala Ala 530 535 540 545 cta cta aaa gac agc aca gca aaa gaggta cta aaa tcc gat aaa ttt 1857 Leu Leu Lys Asp Ser Thr Ala Lys Glu ValLeu Lys Ser Asp Lys Phe 550 555 560 aaa gat gca ata act ggt gct ggt aaggac gca cta aaa gag ata ctt 1905 Lys Asp Ala Ile Thr Gly Ala Gly Lys AspAla Leu Lys Glu Ile Leu 565 570 575 act tgt gat aaa ttt aaa gag gca gtaaca ggc aat ggt aaa gac ata 1953 Thr Cys Asp Lys Phe Lys Glu Ala Val ThrGly Asn Gly Lys Asp Ile 580 585 590 cta aaa ggt ata ctt aca gat agc actggt aaa ttt aaa gag cta ata 2001 Leu Lys Gly Ile Leu Thr Asp Ser Thr GlyLys Phe Lys Glu Leu Ile 595 600 605 gaa agc act ggt aag gat aag cta aaagag att ctt aca gat aac acc 2049 Glu Ser Thr Gly Lys Asp Lys Leu Lys GluIle Leu Thr Asp Asn Thr 610 615 620 625 ggt aac ttt aaa ttt ctt gta gaaggc gcc ggt aag gat gaa gca aaa 2097 Gly Asn Phe Lys Phe Leu Val Glu GlyAla Gly Lys Asp Glu Ala Lys 630 635 640 gca gta ctt act cac gag aaa tttaaa gac ttg ttt aat gtc aaa aca 2145 Ala Val Leu Thr His Glu Lys Phe LysAsp Leu Phe Asn Val Lys Thr 645 650 655 aca gct ggc tac gtg aaa gaa atactt acc agc gac aag ttt aaa gaa 2193 Thr Ala Gly Tyr Val Lys Glu Ile LeuThr Ser Asp Lys Phe Lys Glu 660 665 670 ctg ttt act gat gca aca aaa gctggc tac gtg aaa gaa ata ctc acg 2241 Leu Phe Thr Asp Ala Thr Lys Ala GlyTyr Val Lys Glu Ile Leu Thr 675 680 685 aac gat aca gct aag gaa ata cttaca gat caa aca gct aaa gaa gtc 2289 Asn Asp Thr Ala Lys Glu Ile Leu ThrAsp Gln Thr Ala Lys Glu Val 690 695 700 705 cta aag gat ggt aca gct aaagac ata tta aag gac aca aac gca cgt 2337 Leu Lys Asp Gly Thr Ala Lys AspIle Leu Lys Asp Thr Asn Ala Arg 710 715 720 gcg cta cta aaa gac agc acagcc aaa gaa gta cta aaa tgc gat aaa 2385 Ala Leu Leu Lys Asp Ser Thr AlaLys Glu Val Leu Lys Cys Asp Lys 725 730 735 ttt aag gaa gca ata aca ggtgcc ggt aaa gat gag cta aaa tac ata 2433 Phe Lys Glu Ala Ile Thr Gly AlaGly Lys Asp Glu Leu Lys Tyr Ile 740 745 750 ctc act aat agc gag ttt aaaagc tta ttt cat agc aaa gat agc gct 2481 Leu Thr Asn Ser Glu Phe Lys SerLeu Phe His Ser Lys Asp Ser Ala 755 760 765 gaa gct gtt aaa gca ata tttacc cac aat aag ttt aaa gaa cta ctt 2529 Glu Ala Val Lys Ala Ile Phe ThrHis Asn Lys Phe Lys Glu Leu Leu 770 775 780 785 gaa cat gca aga aca acccaa aca ata cgc agg cgc ttt gca aat gct 2577 Glu His Ala Arg Thr Thr GlnThr Ile Arg Arg Arg Phe Ala Asn Ala 790 795 800 tta gat caa cta aaa gcgcta att acc tgt ggc agc ggt gat cat gca 2625 Leu Asp Gln Leu Lys Ala LeuIle Thr Cys Gly Ser Gly Asp His Ala 805 810 815 aca aaa cta caa gcc tttgga agt gca cta tgc acc aaa aag aag gag 2673 Thr Lys Leu Gln Ala Phe GlySer Ala Leu Cys Thr Lys Lys Lys Glu 820 825 830 ttg tgc agt aat ttt agctgt gca aac tgc agt agt aca aca act gca 2721 Leu Cys Ser Asn Phe Ser CysAla Asn Cys Ser Ser Thr Thr Thr Ala 835 840 845 taattacgta gcgctaggtgggggtaattt acccccacct agctagaatc acacggggaa 2781 ctttctctct attactagggtcttaggatt tacaaacaaa ttactatgac agcca 2836 4 849 PRT Ehrlichia risticii4 Met Ser Asn Glu Thr Leu Leu Ser Val Leu Ser Asp Glu Thr His Phe 1 5 1015 Ala Asn Leu Val Asp Glu Leu Leu Leu Ile Leu Val Lys Asp Ser Ile 20 2530 Phe Thr Gln Val Ile Lys Gly Glu Gly Lys Thr Glu Leu Lys Asp Ile 35 4045 Leu Thr Asp Asn Thr Gly Lys Phe Lys Glu Leu Ile Glu Ser Ala Gly 50 5560 Lys Asp Ile Leu Lys Glu Ile Leu Thr Asp Asn Thr Gly Asn Phe Lys 65 7075 80 Gly Leu Ile Glu Gly Asn Gly Lys Thr Glu Ala Lys Glu Val Arg Thr 8590 95 Asn Glu Lys Phe Lys Glu Leu Phe Gly Ser Asn Gly Lys Asp Ile Leu100 105 110 Lys Asp Ile Leu Thr Asp Asn Thr Gly Asn Phe Lys Gly Leu IleGlu 115 120 125 Ser Ala Ala Lys Gly Lys Leu Lys Asp Leu Leu Ile Asp GluLys Phe 130 135 140 Gln Lys Leu Phe Glu Asp Glu Thr Lys Ala Gly Arg ValLys Glu Ile 145 150 155 160 Leu Thr Asp Ser Asn Ala Lys Glu Ile Leu ThrAsn Glu Val Ala Lys 165 170 175 Glu Val Leu Lys Ser Asp Lys Phe Lys GluAla Ile Thr Gly Asp Gly 180 185 190 Lys Asp Ala Leu Lys Glu Ile Leu ThrCys Asp Lys Phe Lys Glu Ala 195 200 205 Val Thr Gly Asn Gly Lys Asp IleLeu Lys Gly Ile Leu Thr Asp Ser 210 215 220 Thr Gly Lys Phe Lys Glu LeuIle Glu Ser Thr Ser Lys Asp Ile Leu 225 230 235 240 Lys Glu Ile Leu ThrAsp Asn Thr Gly Asn Phe Lys Gly Leu Ile Glu 245 250 255 Ser Thr Gly LysGlu Lys Val Lys Glu Leu Leu Ile Asp Gly Lys Phe 260 265 270 Lys Asp LeuPhe Thr Asp Ala Thr Lys Ala Gly Tyr Val Lys Glu Ile 275 280 285 Leu ThrAsn Asp Thr Ala Lys Glu Val Leu Thr Asp Gln Thr Ala Lys 290 295 300 GluVal Leu Lys Asp Ser Thr Ala Lys Asp Ile Leu Lys Asp Thr Asn 305 310 315320 Ala Ala Ala Val Leu Lys Asn Ser Thr Ala Lys Glu Ile Leu Thr Asn 325330 335 Gln Thr Ala Lys Glu Val Leu Thr Asp Gly Thr Ser Lys Glu Val Leu340 345 350 Lys Glu Ile Leu Thr Cys Asp Lys Phe Lys Glu Ala Val Thr GlyAsp 355 360 365 Gly Lys Asp Ile Leu Lys Gly Ile Leu Thr Asp Ser Thr GlyLys Phe 370 375 380 Lys Glu Leu Ile Glu Ser Thr Gly Lys Asp Ile Leu LysAsp Ile Leu 385 390 395 400 Thr Asp Ser Thr Gly Lys Phe Lys Glu Leu IleGlu Val Leu Val Lys 405 410 415 Asn Lys Leu Lys Glu Ile Leu Thr Asp AsnThr Gly Asn Phe Lys Gly 420 425 430 Leu Val Glu Gly Ala Gly Lys Asp GluAla Lys Ala Val Leu Thr Asp 435 440 445 Glu Lys Phe Lys Gly Leu Phe AspAsp Lys Thr Ile Ala Gly Tyr Val 450 455 460 Lys Glu Ile Leu Thr Ser GluLys Phe Lys Lys Leu Phe Glu Ser Ala 465 470 475 480 Gly Lys Thr Lys ValLys Glu Leu Leu Ile Asp Glu Lys Phe Gln Lys 485 490 495 Leu Phe Glu AspAsp Thr Lys Ala Ser His Val Lys Glu Ile Leu Thr 500 505 510 Asn Asp ThrAla Lys Glu Ile Leu Thr Asp Gln Thr Ala Lys Glu Val 515 520 525 Leu LysAsp Ser Thr Ala Lys Glu Ile Leu Lys Asp Thr Asn Ala Ala 530 535 540 AlaLeu Leu Lys Asp Ser Thr Ala Lys Glu Val Leu Lys Ser Asp Lys 545 550 555560 Phe Lys Asp Ala Ile Thr Gly Ala Gly Lys Asp Ala Leu Lys Glu Ile 565570 575 Leu Thr Cys Asp Lys Phe Lys Glu Ala Val Thr Gly Asn Gly Lys Asp580 585 590 Ile Leu Lys Gly Ile Leu Thr Asp Ser Thr Gly Lys Phe Lys GluLeu 595 600 605 Ile Glu Ser Thr Gly Lys Asp Lys Leu Lys Glu Ile Leu ThrAsp Asn 610 615 620 Thr Gly Asn Phe Lys Phe Leu Val Glu Gly Ala Gly LysAsp Glu Ala 625 630 635 640 Lys Ala Val Leu Thr His Glu Lys Phe Lys AspLeu Phe Asn Val Lys 645 650 655 Thr Thr Ala Gly Tyr Val Lys Glu Ile LeuThr Ser Asp Lys Phe Lys 660 665 670 Glu Leu Phe Thr Asp Ala Thr Lys AlaGly Tyr Val Lys Glu Ile Leu 675 680 685 Thr Asn Asp Thr Ala Lys Glu IleLeu Thr Asp Gln Thr Ala Lys Glu 690 695 700 Val Leu Lys Asp Gly Thr AlaLys Asp Ile Leu Lys Asp Thr Asn Ala 705 710 715 720 Arg Ala Leu Leu LysAsp Ser Thr Ala Lys Glu Val Leu Lys Cys Asp 725 730 735 Lys Phe Lys GluAla Ile Thr Gly Ala Gly Lys Asp Glu Leu Lys Tyr 740 745 750 Ile Leu ThrAsn Ser Glu Phe Lys Ser Leu Phe His Ser Lys Asp Ser 755 760 765 Ala GluAla Val Lys Ala Ile Phe Thr His Asn Lys Phe Lys Glu Leu 770 775 780 LeuGlu His Ala Arg Thr Thr Gln Thr Ile Arg Arg Arg Phe Ala Asn 785 790 795800 Ala Leu Asp Gln Leu Lys Ala Leu Ile Thr Cys Gly Ser Gly Asp His 805810 815 Ala Thr Lys Leu Gln Ala Phe Gly Ser Ala Leu Cys Thr Lys Lys Lys820 825 830 Glu Leu Cys Ser Asn Phe Ser Cys Ala Asn Cys Ser Ser Thr ThrThr 835 840 845 Ala 5 1937 DNA Ehrlichia risticii CDS (175)..(1791) 5attggatcta aataatatac actggaggtt cgtattttct attatgaaag ggatagaatg 60ttaaatttta tgatttttta taataaaaat agatataaaa tttagtaatt ttataaattt 120tttataacaa aggactaccc tccctacata aaatttctaa gacgaaaaat ccct atg 177 Met1 tca aat gaa aca ctt ctg agc gta ctt tct gat gaa acg cac ttt gct 225Ser Asn Glu Thr Leu Leu Ser Val Leu Ser Asp Glu Thr His Phe Ala 5 10 15aat cta gtt gat gaa ctt ctt ctc agc ttg gtt aaa gac agt att ttc 273 AsnLeu Val Asp Glu Leu Leu Leu Ser Leu Val Lys Asp Ser Ile Phe 20 25 30 actcaa gta ata aaa ggc gag gga aag aca gaa tta aaa gac att ctt 321 Thr GlnVal Ile Lys Gly Glu Gly Lys Thr Glu Leu Lys Asp Ile Leu 35 40 45 aca gatagc act ggc aag ttt aaa gag ctg ata gga agt agc ggt aag 369 Thr Asp SerThr Gly Lys Phe Lys Glu Leu Ile Gly Ser Ser Gly Lys 50 55 60 65 gat atacta aaa agc ata cac aca gat ggc tca ggc aac ttt aaa ggc 417 Asp Ile LeuLys Ser Ile His Thr Asp Gly Ser Gly Asn Phe Lys Gly 70 75 80 ctt ata caaagc aca ggt aag gca gaa gta aaa gag gta ctc act aat 465 Leu Ile Gln SerThr Gly Lys Ala Glu Val Lys Glu Val Leu Thr Asn 85 90 95 gaa aaa ttc aaagag ctt ttt gga agc gaa ggt aaa gac ata cta aaa 513 Glu Lys Phe Lys GluLeu Phe Gly Ser Glu Gly Lys Asp Ile Leu Lys 100 105 110 gag ata ctt acagac aat acc ggc aat ttt aaa ggg ctt ata gaa ggc 561 Glu Ile Leu Thr AspAsn Thr Gly Asn Phe Lys Gly Leu Ile Glu Gly 115 120 125 aaa ggt aag gatgaa gca aag gga gta ctt act gac gag aaa ttt aaa 609 Lys Gly Lys Asp GluAla Lys Gly Val Leu Thr Asp Glu Lys Phe Lys 130 135 140 145 ggc ttg tttgat gac aaa aca ata gct ggc tat gta aaa gaa ata ctc 657 Gly Leu Phe AspAsp Lys Thr Ile Ala Gly Tyr Val Lys Glu Ile Leu 150 155 160 acc agc gagagt tta aaa aac tgt ttg aaa ggt gca ggt aag act aaa 705 Thr Ser Glu SerLeu Lys Asn Cys Leu Lys Gly Ala Gly Lys Thr Lys 165 170 175 gta aaa gaactc ctc att gat gag aag ttt caa aaa tta ttt gag gat 753 Val Lys Glu LeuLeu Ile Asp Glu Lys Phe Gln Lys Leu Phe Glu Asp 180 185 190 gac acg aaagcc agt cat gta aaa gaa ata ctt aca gac agt aac gct 801 Asp Thr Lys AlaSer His Val Lys Glu Ile Leu Thr Asp Ser Asn Ala 195 200 205 aag gaa atactc aca aat gaa gta gca aaa gag gta cta aaa tcc gat 849 Lys Glu Ile LeuThr Asn Glu Val Ala Lys Glu Val Leu Lys Ser Asp 210 215 220 225 aaa tttaaa gat gca ata act ggt gct ggt aag gac gca cta aaa gag 897 Lys Phe LysAsp Ala Ile Thr Gly Ala Gly Lys Asp Ala Leu Lys Glu 230 235 240 ata cttact tgc gat aaa ttt aaa gat gca gta aca ggt aat ggt aag 945 Ile Leu ThrCys Asp Lys Phe Lys Asp Ala Val Thr Gly Asn Gly Lys 245 250 255 gac gcacta aaa gaa ata ctt act tgc gat aaa ttt aaa gat gca gta 993 Asp Ala LeuLys Glu Ile Leu Thr Cys Asp Lys Phe Lys Asp Ala Val 260 265 270 aca ggcaat ggt aaa gac aag cta aaa gag att ctt act cac gag aag 1041 Thr Gly AsnGly Lys Asp Lys Leu Lys Glu Ile Leu Thr His Glu Lys 275 280 285 ttt aaagca ctc ata gag agt gaa ggc aaa gac ata ctg aaa gaa att 1089 Phe Lys AlaLeu Ile Glu Ser Glu Gly Lys Asp Ile Leu Lys Glu Ile 290 295 300 305 cttaca gat agt acc ggt aaa ttt aaa gag cta ata gaa agc act ggt 1137 Leu ThrAsp Ser Thr Gly Lys Phe Lys Glu Leu Ile Glu Ser Thr Gly 310 315 320 aaagac aag cta aaa gag att ttc aca gat aac acc ggt aac ttt aaa 1185 Lys AspLys Leu Lys Glu Ile Phe Thr Asp Asn Thr Gly Asn Phe Lys 325 330 335 gggctt gta gaa ggc gcc ggt aag gat gaa gca aaa gca gta ctt act 1233 Gly LeuVal Glu Gly Ala Gly Lys Asp Glu Ala Lys Ala Val Leu Thr 340 345 350 cacgag aaa ttt aaa gac ttg ttt aat gac aaa aca aca gct ggc tac 1281 His GluLys Phe Lys Asp Leu Phe Asn Asp Lys Thr Thr Ala Gly Tyr 355 360 365 gtgaaa gaa ata ctc acc agt gat aag ttt aaa aaa tta ttt gag gac 1329 Val LysGlu Ile Leu Thr Ser Asp Lys Phe Lys Lys Leu Phe Glu Asp 370 375 380 385aat acc aaa gct ggc tac gtg aaa gaa ata ctc acg aac gat aca gct 1377 AsnThr Lys Ala Gly Tyr Val Lys Glu Ile Leu Thr Asn Asp Thr Ala 390 395 400aag gaa ata ctc aca aat caa aca gct aaa gaa gtc cta aaa gac agc 1425 LysGlu Ile Leu Thr Asn Gln Thr Ala Lys Glu Val Leu Lys Asp Ser 405 410 415aca gcc aaa gaa ata cta aaa tgc gat aaa ttt aag gac gca ata aca 1473 ThrAla Lys Glu Ile Leu Lys Cys Asp Lys Phe Lys Asp Ala Ile Thr 420 425 430ggc gct ggt aaa gat gag cta aaa tac ata ctc act aat aac gag ttt 1521 GlyAla Gly Lys Asp Glu Leu Lys Tyr Ile Leu Thr Asn Asn Glu Phe 435 440 445aaa agc tta ttt gat agc aaa gat agc gct gaa gct gtt aaa gca ata 1569 LysSer Leu Phe Asp Ser Lys Asp Ser Ala Glu Ala Val Lys Ala Ile 450 455 460465 ttt acc cac aat aag ttt aaa gaa cta ctt aaa acg tgc aag gac aac 1617Phe Thr His Asn Lys Phe Lys Glu Leu Leu Lys Thr Cys Lys Asp Asn 470 475480 cca aaa aat acg gcg gcg ctt gca gct gct tta gat gaa cta aaa gat 1665Pro Lys Asn Thr Ala Ala Leu Ala Ala Ala Leu Asp Glu Leu Lys Asp 485 490495 cta att acg tgt gac cgc aat aat cat gca aca aaa cta caa gcc ttt 1713Leu Ile Thr Cys Asp Arg Asn Asn His Ala Thr Lys Leu Gln Ala Phe 500 505510 gga agt gca cta tgc acc aga aaa aaa gag tcg tgc gat aat ttt agc 1761Gly Ser Ala Leu Cys Thr Arg Lys Lys Glu Ser Cys Asp Asn Phe Ser 515 520525 cct gca agc tgc agt agt aca gca gct aca taattacgta gcgctaggtg 1811Pro Ala Ser Cys Ser Ser Thr Ala Ala Thr 530 535 ggggtaaatt acccccacctacgtagaatc acacggggaa ctttctctct attactgagg 1871 tcttaggatt tactttcaaattactatgac agccgattaa attattatga cagacgatac 1931 actttt 1937 6 539 PRTEhrlichia risticii 6 Met Ser Asn Glu Thr Leu Leu Ser Val Leu Ser Asp GluThr His Phe 1 5 10 15 Ala Asn Leu Val Asp Glu Leu Leu Leu Ser Leu ValLys Asp Ser Ile 20 25 30 Phe Thr Gln Val Ile Lys Gly Glu Gly Lys Thr GluLeu Lys Asp Ile 35 40 45 Leu Thr Asp Ser Thr Gly Lys Phe Lys Glu Leu IleGly Ser Ser Gly 50 55 60 Lys Asp Ile Leu Lys Ser Ile His Thr Asp Gly SerGly Asn Phe Lys 65 70 75 80 Gly Leu Ile Gln Ser Thr Gly Lys Ala Glu ValLys Glu Val Leu Thr 85 90 95 Asn Glu Lys Phe Lys Glu Leu Phe Gly Ser GluGly Lys Asp Ile Leu 100 105 110 Lys Glu Ile Leu Thr Asp Asn Thr Gly AsnPhe Lys Gly Leu Ile Glu 115 120 125 Gly Lys Gly Lys Asp Glu Ala Lys GlyVal Leu Thr Asp Glu Lys Phe 130 135 140 Lys Gly Leu Phe Asp Asp Lys ThrIle Ala Gly Tyr Val Lys Glu Ile 145 150 155 160 Leu Thr Ser Glu Ser LeuLys Asn Cys Leu Lys Gly Ala Gly Lys Thr 165 170 175 Lys Val Lys Glu LeuLeu Ile Asp Glu Lys Phe Gln Lys Leu Phe Glu 180 185 190 Asp Asp Thr LysAla Ser His Val Lys Glu Ile Leu Thr Asp Ser Asn 195 200 205 Ala Lys GluIle Leu Thr Asn Glu Val Ala Lys Glu Val Leu Lys Ser 210 215 220 Asp LysPhe Lys Asp Ala Ile Thr Gly Ala Gly Lys Asp Ala Leu Lys 225 230 235 240Glu Ile Leu Thr Cys Asp Lys Phe Lys Asp Ala Val Thr Gly Asn Gly 245 250255 Lys Asp Ala Leu Lys Glu Ile Leu Thr Cys Asp Lys Phe Lys Asp Ala 260265 270 Val Thr Gly Asn Gly Lys Asp Lys Leu Lys Glu Ile Leu Thr His Glu275 280 285 Lys Phe Lys Ala Leu Ile Glu Ser Glu Gly Lys Asp Ile Leu LysGlu 290 295 300 Ile Leu Thr Asp Ser Thr Gly Lys Phe Lys Glu Leu Ile GluSer Thr 305 310 315 320 Gly Lys Asp Lys Leu Lys Glu Ile Phe Thr Asp AsnThr Gly Asn Phe 325 330 335 Lys Gly Leu Val Glu Gly Ala Gly Lys Asp GluAla Lys Ala Val Leu 340 345 350 Thr His Glu Lys Phe Lys Asp Leu Phe AsnAsp Lys Thr Thr Ala Gly 355 360 365 Tyr Val Lys Glu Ile Leu Thr Ser AspLys Phe Lys Lys Leu Phe Glu 370 375 380 Asp Asn Thr Lys Ala Gly Tyr ValLys Glu Ile Leu Thr Asn Asp Thr 385 390 395 400 Ala Lys Glu Ile Leu ThrAsn Gln Thr Ala Lys Glu Val Leu Lys Asp 405 410 415 Ser Thr Ala Lys GluIle Leu Lys Cys Asp Lys Phe Lys Asp Ala Ile 420 425 430 Thr Gly Ala GlyLys Asp Glu Leu Lys Tyr Ile Leu Thr Asn Asn Glu 435 440 445 Phe Lys SerLeu Phe Asp Ser Lys Asp Ser Ala Glu Ala Val Lys Ala 450 455 460 Ile PheThr His Asn Lys Phe Lys Glu Leu Leu Lys Thr Cys Lys Asp 465 470 475 480Asn Pro Lys Asn Thr Ala Ala Leu Ala Ala Ala Leu Asp Glu Leu Lys 485 490495 Asp Leu Ile Thr Cys Asp Arg Asn Asn His Ala Thr Lys Leu Gln Ala 500505 510 Phe Gly Ser Ala Leu Cys Thr Arg Lys Lys Glu Ser Cys Asp Asn Phe515 520 525 Ser Pro Ala Ser Cys Ser Ser Thr Ala Ala Thr 530 535 7 1791DNA Ehrlichia risticii CDS (175)..(1677) 7 attggatcta aataatgtacactggaggtt cgtattttct attatgaaag ggatagaatg 60 ttaaatttta tgattttttataataaaaat agatataaaa tttagtagtt ttataaattt 120 ttcataacaa aggactatcctccttgcata aaatttctaa gacgaaaaat cctt atg 177 Met 1 tca aat gaa aca cttctg agc gta ctt tct gat gaa acg cac ttt gct 225 Ser Asn Glu Thr Leu LeuSer Val Leu Ser Asp Glu Thr His Phe Ala 5 10 15 aat cta gtt gat gaa cttctt ctc agc ttg gtt aaa gac agt att ttc 273 Asn Leu Val Asp Glu Leu LeuLeu Ser Leu Val Lys Asp Ser Ile Phe 20 25 30 act caa gta ata aaa ggc gaggga aag aca gaa tta aaa gac att ctt 321 Thr Gln Val Ile Lys Gly Glu GlyLys Thr Glu Leu Lys Asp Ile Leu 35 40 45 aca gat agc act ggc aag ttt aaagag ctg ata gga agt agc ggt aag 369 Thr Asp Ser Thr Gly Lys Phe Lys GluLeu Ile Gly Ser Ser Gly Lys 50 55 60 65 gat ata cta aaa agc ata ctc acagat ggc tca ggc aac ttt aaa ggc 417 Asp Ile Leu Lys Ser Ile Leu Thr AspGly Ser Gly Asn Phe Lys Gly 70 75 80 ctt ata caa agc aca ggt aag gca gaagta aaa gag gta ctc act aat 465 Leu Ile Gln Ser Thr Gly Lys Ala Glu ValLys Glu Val Leu Thr Asn 85 90 95 gaa aaa ttc aaa gag ctt ttt gga agc gatggt aag gat ata tta aaa 513 Glu Lys Phe Lys Glu Leu Phe Gly Ser Asp GlyLys Asp Ile Leu Lys 100 105 110 gac ata ctc aca gat agc act ggt aag tttaaa gag ctg ata gga agt 561 Asp Ile Leu Thr Asp Ser Thr Gly Lys Phe LysGlu Leu Ile Gly Ser 115 120 125 agc ggt aag gac ata cta aaa aac att cttaca gat agc acc ggt aag 609 Ser Gly Lys Asp Ile Leu Lys Asn Ile Leu ThrAsp Ser Thr Gly Lys 130 135 140 145 ttt aaa gaa ctt ata gaa agt gca ggtaag ggt aag ctg aaa gac ctt 657 Phe Lys Glu Leu Ile Glu Ser Ala Gly LysGly Lys Leu Lys Asp Leu 150 155 160 ctt att gat gga aac ttt aaa aaa ttattt gag gat gac acg aaa gct 705 Leu Ile Asp Gly Asn Phe Lys Lys Leu PheGlu Asp Asp Thr Lys Ala 165 170 175 gct cat gta aaa gaa ata ctt aca gacagc aac gct aag gaa ata ctc 753 Ala His Val Lys Glu Ile Leu Thr Asp SerAsn Ala Lys Glu Ile Leu 180 185 190 aca aat gaa gta gca aaa gag gta ctaaaa tcc gat aaa ttt aaa gat 801 Thr Asn Glu Val Ala Lys Glu Val Leu LysSer Asp Lys Phe Lys Asp 195 200 205 gca ata act ggt gct ggt aag gac gcacta aaa gag ata ctt act tgc 849 Ala Ile Thr Gly Ala Gly Lys Asp Ala LeuLys Glu Ile Leu Thr Cys 210 215 220 225 gat aaa ttt aaa gat gca gta acaggc aat ggt aag gac gca cta aaa 897 Asp Lys Phe Lys Asp Ala Val Thr GlyAsn Gly Lys Asp Ala Leu Lys 230 235 240 gaa ata ctt act tgc gat aaa tttaaa gat gca gta aca ggc aat ggt 945 Glu Ile Leu Thr Cys Asp Lys Phe LysAsp Ala Val Thr Gly Asn Gly 245 250 255 aaa gac aag cta aaa gag att cttact cac gag aag ttt aaa gca ctc 993 Lys Asp Lys Leu Lys Glu Ile Leu ThrHis Glu Lys Phe Lys Ala Leu 260 265 270 ata gag agt gaa ggc aaa gac atactg aaa gac att ctt aca gat agt 1041 Ile Glu Ser Glu Gly Lys Asp Ile LeuLys Asp Ile Leu Thr Asp Ser 275 280 285 acc ggt aaa ttt aaa gag cta atagaa agc acg ggt aag gat gaa gca 1089 Thr Gly Lys Phe Lys Glu Leu Ile GluSer Thr Gly Lys Asp Glu Ala 290 295 300 305 aaa gca gta ctt act gac gagaaa ttt aaa gac ttg ttt aat gac aaa 1137 Lys Ala Val Leu Thr Asp Glu LysPhe Lys Asp Leu Phe Asn Asp Lys 310 315 320 aca aca gct ggc tac gtg aaagaa ata ctc acc agt gat aag ttt aaa 1185 Thr Thr Ala Gly Tyr Val Lys GluIle Leu Thr Ser Asp Lys Phe Lys 325 330 335 aaa tta ttt gag gac aat accaaa gct ggc tac gtg aaa gaa ata ctc 1233 Lys Leu Phe Glu Asp Asn Thr LysAla Gly Tyr Val Lys Glu Ile Leu 340 345 350 acg aac gat aca gct aag gaaata ctt acc aat cat aaa ttt aag gaa 1281 Thr Asn Asp Thr Ala Lys Glu IleLeu Thr Asn His Lys Phe Lys Glu 355 360 365 gca ata act ggc gat ggt aaagac ata ctg aaa gaa att ctt aca gat 1329 Ala Ile Thr Gly Asp Gly Lys AspIle Leu Lys Glu Ile Leu Thr Asp 370 375 380 385 agc act ggt aac ttt aaaggc gca ata aca ggt gcc ggt aaa gat cag 1377 Ser Thr Gly Asn Phe Lys GlyAla Ile Thr Gly Ala Gly Lys Asp Gln 390 395 400 cta aaa tac ata ctc actaat agc gag ttt aaa agc tta ttt gat agc 1425 Leu Lys Tyr Ile Leu Thr AsnSer Glu Phe Lys Ser Leu Phe Asp Ser 405 410 415 aaa gat agc gct gaa gctgtt aaa gaa ata ttt acc cac agt aag ttt 1473 Lys Asp Ser Ala Glu Ala ValLys Glu Ile Phe Thr His Ser Lys Phe 420 425 430 aaa gaa cta ctt aaa acgtgc aag gac aac cca aaa aat acg gcg gcg 1521 Lys Glu Leu Leu Lys Thr CysLys Asp Asn Pro Lys Asn Thr Ala Ala 435 440 445 ctt gca gct gct tta gatgaa cta aaa gat cta att acc tgt ggc agc 1569 Leu Ala Ala Ala Leu Asp GluLeu Lys Asp Leu Ile Thr Cys Gly Ser 450 455 460 465 ggt gat cat gca acaaaa cta caa gcc ttt gga agt gca cta tgc acc 1617 Gly Asp His Ala Thr LysLeu Gln Ala Phe Gly Ser Ala Leu Cys Thr 470 475 480 aga aaa aaa gag tcgtgc gat aat ttt agc tct gca aac tgc agt agt 1665 Arg Lys Lys Glu Ser CysAsp Asn Phe Ser Ser Ala Asn Cys Ser Ser 485 490 495 aca aca act gcataattacgta gcgctaggtg ggggtaattt acccccacct 1717 Thr Thr Thr Ala 500agctagaatc acacggggaa ctttctctct attactaggg tcttaggatt acaaacaaat 1777tactatgaca gcca 1791 8 501 PRT Ehrlichia risticii 8 Met Ser Asn Glu ThrLeu Leu Ser Val Leu Ser Asp Glu Thr His Phe 1 5 10 15 Ala Asn Leu ValAsp Glu Leu Leu Leu Ser Leu Val Lys Asp Ser Ile 20 25 30 Phe Thr Gln ValIle Lys Gly Glu Gly Lys Thr Glu Leu Lys Asp Ile 35 40 45 Leu Thr Asp SerThr Gly Lys Phe Lys Glu Leu Ile Gly Ser Ser Gly 50 55 60 Lys Asp Ile LeuLys Ser Ile Leu Thr Asp Gly Ser Gly Asn Phe Lys 65 70 75 80 Gly Leu IleGln Ser Thr Gly Lys Ala Glu Val Lys Glu Val Leu Thr 85 90 95 Asn Glu LysPhe Lys Glu Leu Phe Gly Ser Asp Gly Lys Asp Ile Leu 100 105 110 Lys AspIle Leu Thr Asp Ser Thr Gly Lys Phe Lys Glu Leu Ile Gly 115 120 125 SerSer Gly Lys Asp Ile Leu Lys Asn Ile Leu Thr Asp Ser Thr Gly 130 135 140Lys Phe Lys Glu Leu Ile Glu Ser Ala Gly Lys Gly Lys Leu Lys Asp 145 150155 160 Leu Leu Ile Asp Gly Asn Phe Lys Lys Leu Phe Glu Asp Asp Thr Lys165 170 175 Ala Ala His Val Lys Glu Ile Leu Thr Asp Ser Asn Ala Lys GluIle 180 185 190 Leu Thr Asn Glu Val Ala Lys Glu Val Leu Lys Ser Asp LysPhe Lys 195 200 205 Asp Ala Ile Thr Gly Ala Gly Lys Asp Ala Leu Lys GluIle Leu Thr 210 215 220 Cys Asp Lys Phe Lys Asp Ala Val Thr Gly Asn GlyLys Asp Ala Leu 225 230 235 240 Lys Glu Ile Leu Thr Cys Asp Lys Phe LysAsp Ala Val Thr Gly Asn 245 250 255 Gly Lys Asp Lys Leu Lys Glu Ile LeuThr His Glu Lys Phe Lys Ala 260 265 270 Leu Ile Glu Ser Glu Gly Lys AspIle Leu Lys Asp Ile Leu Thr Asp 275 280 285 Ser Thr Gly Lys Phe Lys GluLeu Ile Glu Ser Thr Gly Lys Asp Glu 290 295 300 Ala Lys Ala Val Leu ThrAsp Glu Lys Phe Lys Asp Leu Phe Asn Asp 305 310 315 320 Lys Thr Thr AlaGly Tyr Val Lys Glu Ile Leu Thr Ser Asp Lys Phe 325 330 335 Lys Lys LeuPhe Glu Asp Asn Thr Lys Ala Gly Tyr Val Lys Glu Ile 340 345 350 Leu ThrAsn Asp Thr Ala Lys Glu Ile Leu Thr Asn His Lys Phe Lys 355 360 365 GluAla Ile Thr Gly Asp Gly Lys Asp Ile Leu Lys Glu Ile Leu Thr 370 375 380Asp Ser Thr Gly Asn Phe Lys Gly Ala Ile Thr Gly Ala Gly Lys Asp 385 390395 400 Gln Leu Lys Tyr Ile Leu Thr Asn Ser Glu Phe Lys Ser Leu Phe Asp405 410 415 Ser Lys Asp Ser Ala Glu Ala Val Lys Glu Ile Phe Thr His SerLys 420 425 430 Phe Lys Glu Leu Leu Lys Thr Cys Lys Asp Asn Pro Lys AsnThr Ala 435 440 445 Ala Leu Ala Ala Ala Leu Asp Glu Leu Lys Asp Leu IleThr Cys Gly 450 455 460 Ser Gly Asp His Ala Thr Lys Leu Gln Ala Phe GlySer Ala Leu Cys 465 470 475 480 Thr Arg Lys Lys Glu Ser Cys Asp Asn PheSer Ser Ala Asn Cys Ser 485 490 495 Ser Thr Thr Thr Ala 500 9 33 DNAArtificial Sequence Description of Artificial Sequence primer 9cataaaattt ctaagacgaa ggatccctat gtc 33 10 33 DNA Artificial SequenceDescription of Artificial Sequence primer 10 gagagaaagt tccccgtgtgaattctagct agg 33 11 18 DNA Artificial Sequence Description ofArtificial Sequence primer 11 atactaaaaa gcatactc 18 12 18 DNAArtificial Sequence Description of Artificial Sequence primer 12ttctacaagc cctttaaa 18 13 18 DNA Artificial Sequence Description ofArtificial Sequence primer 13 gaatgttcag ctttccgg 18 14 18 DNAArtificial Sequence Description of Artificial Sequence primer 14agctgtatcg ttcgtgag 18 15 19 DNA Artificial Sequence Description ofArtificial Sequence primer 15 gtatacttac agatagcac 19 16 19 DNAArtificial Sequence Description of Artificial Sequence primer 16gccgacagta tcattaaac 19 17 11 DNA Artificial Sequence 17 aaagaaatac t 1118 11 DNA Ehrlichia risticii 18 gaaatactca c 11 19 11 DNA Ehrlichiaristicii 19 aaatttaaag a 11 20 11 DNA Ehrlichia risticii 20 ctaaaagaga t11 21 11 DNA Ehrlichia risticii 21 aaagacatac t 11 22 11 DNA Ehrlichiaristicii 22 tttaaagagc t 11 23 11 DNA Ehrlichia risticii 23 attttttata a11 24 11 DNA Ehrlichia risticii 24 aactttaaag g 11 25 11 DNA Ehrlichiaristicii 25 aagtttaaag a 11 26 11 DNA Ehrlichia risticii 26 tactcactaa t11 27 11 DNA Ehrlichia risticii 27 agtttaaaaa a 11 28 11 DNA Ehrlichiaristicii 28 ataagtttaa a 11 29 11 DNA Ehrlichia risticii 29 atacttacag a11 30 11 DNA Ehrlichia risticii 30 aaatttaaag a 11 31 11 DNA Ehrlichiaristicii 31 ctaaaagaga t 11 32 11 DNA Ehrlichia risticii 32 aaagaaatac t11 33 11 DNA Ehrlichia risticii 33 tacttacaga t 11 34 11 DNA Ehrlichiaristicii 34 aaagacatac t 11 35 11 DNA Ehrlichia risticii 35 acagctaaag a11 36 11 DNA Ehrlichia risticii 36 tttaaagaac t 11 37 11 DNA Ehrlichiaristicii 37 gaaatactta c 11 38 11 DNA Ehrlichia risticii 38 agcactggta a11 39 11 DNA Ehrlichia risticii 39 gataaattta a 11 40 11 DNA Ehrlichiaristicii 40 cttatagaaa g 11 41 11 DNA Ehrlichia risticii 41 gaaatactca c11 42 11 DNA Ehrlichia risticii 42 accggtaact t 11 43 11 DNA Ehrlichiaristicii 43 atgcaacaaa a 11 44 11 DNA Ehrlichia risticii 44 gctaaagaag t11 45 11 DNA Ehrlichia risticii 45 cttacagata a 11 46 11 DNA Ehrlichiaristicii 46 gcaataactg g 11 47 11 DNA Ehrlichia risticii 47 atggtaagga c11 48 11 DNA Ehrlichia risticii 48 acttatagaa g 11

1. An isolated and purified antigen which is expressed by a wild-type E.risticii strain and is specific to the wild-type E. risticii strain. 2.The antigen of claim 1, which stimulates a protective immune responseagainst infection by the wild-type E. risticii strain when a host isimmunized with the antigen.
 3. The antigen of claim 1, which stimulatesa protective immune response against infection by at least one otherwild-type E. risticii strain when a host is immunized with the antigen.4. The antigen of claim 1, which has a molecular weight of about 40 toabout 90 kDa.
 5. The antigen of claim 1, which is expressed by E.risticii strain 25-D.
 6. The antigen of claim 1, which is expressed byE. risticii strain 90-12.
 7. The antigen of claim 1, which is expressedby ATCC type E. risticii strain.
 8. The antigen of claim 1, whichcomprises the amino acid sequence shown in FIG.
 2. 9. The antigen ofclaim 1, which comprises the amino acid sequence shown in FIG.
 3. 10.The antigen of claim 1, which comprises the amino acid sequence shown inFIG.
 4. 11. An isolated and purified nucleic acid encoding the antigenof claim
 1. 12. An expression vector comprising the nucleic acid ofclaim
 6. 13. A host cell transformed with the expression vector of claim12, wherein the transformed host produces the antigen.
 14. A method ofproduciLng an antigen which is expressed by a wild-type E. risticiistrain and is specific to the wild-type E. risticii strain, comprisingculturing the transformed host cell of claim 13 in a suitable culruremedium, and isolating the antigen.
 15. An immunogenic pharmaceuticalcomposition, comprising the antigen of claim 1 and a pharmaceuticallyacceptable carrier.
 16. A method of inducing an immune response,comprising administering an effective amount of the immunogeniccomposition of claim 15 to a host.
 17. An isolated and purified proteincomprising an amino acid sequence selected from the group consisting ofthe amino acid sequence recited in FIG. 2, the amino acid sequencerecited in FIG. 3 and the amino acid sequence recited in FIG.
 4. 18. Theprotein of claim 17, comprising the amino acid sequence recited in FIG.2.
 19. The protein of claim 17, comprising the amino acid sequencerecited in FIG.
 3. 20. The protein of claim 17, comprising the aminoacid sequence recited in FIG.
 4. 21. The protein of claim 17, whichconsists of said amino acid sequence.
 22. An isolated and purifiednucleic acid encoding the protein of claim
 17. 23. An expression vectorcomprising the nucleic acid of claim
 22. 24. A host cell transformedwith the expression vector of claim 23, wherein the transformed hostproduces the protein.
 25. A method of producing a protein, comprisingculturing the transformed host cell of claim 24 in a suitable culturemedium, and isolating the antigen.
 26. An immunogenic pharmaceuticalcomposition, comprising the protein of claim 17 and a pharmaceuticallyacceptable carrier.
 27. A method of inducing an immune response,comprising administering an effective amount of the immunogeniccomposition of claim 26 to a host.